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  • 751. Wärnmark, A
    et al.
    Wikström, Anja
    KTH.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Gustafsson, J A
    Härd, T
    The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 49, p. 45939-45944Article in journal (Refereed)
    Abstract [en]

    The N-terminal regions of the estrogen receptor ve (ER alpha -N) and beta (ER beta -N) were expressed and purified to homogeneity. Using NAM and circular dichroism spectroscopy, we conclude that both ER alpha -N and ER beta -N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ERa-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ERa-N interacts with TBP, and suggest that structural changes are induced in ERa-N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ER beta -N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ERa-N and ER beta -N. The affinity of the ER alpha -N-TBP interaction was determined to be in the micromolar range (K-D = 10(-6) to 10(-5) m). Our results support models of TBP as a target protein for the N-terminal activation domain of ER alpha. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.

  • 752.
    Wärnmark, Anette
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Intsitute.
    Gustafsson, J A
    Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Architectural principles for the structure and function of the glucocorticoid receptor tau 1 core activation domain2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 20, p. 15014-15018Article in journal (Refereed)
    Abstract [en]

    A 58-amino acid region mediates the core transactivation activity of the glucocorticoid receptor tau 1 activation domain. This tau 1 core domain is unstructured in aqueous buffers, but in the presence of trifluoroethanol three Alpha-helical segments are induced. Two of these putative structural modules have been tested in different combinations with regard to transactivation potential in vivo and binding capacity to the coactivators in vitro, The results show that whereas single modules are not transcriptionally active, any combination of two or three modules is sufficient, with trimodular constructs having the highest activity. However, proteins containing one, two, or three segments bind Ada2 and cAMP-response element-binding protein with similar affinity. A single segment is thus able to bind a target factor but cannot transactivate target genes significantly. The results are consistent with models in which activation domains are comprised of short activation modules that allow multiple interactions with coactivators. Our results also suggest that an increased number of modules may not result in correspondingly higher affinity but instead that the concentration of binding sites is increased, which gives rise to a higher association rate. This is consistent with a model where the association rate for activator-target factor interactions rather than the equilibrium constant is the most relevant measure of activator potency.

  • 753. Wüthrich, Kurt
    et al.
    Güntert, Peter
    Berndt, Kurt D.
    Computer-supported Structure Determination of Proteins in Solution Illustrated with Studies of Protein Proteinase Inhibitors1993In: Innovations in Proteinases and Their Inhibitors / [ed] Avilés, Francese X., Berlin: Walter de Gruyter, 1993, p. 407-Chapter in book (Other academic)
  • 754.
    Xia, Ling
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Björnstedt, M.
    Nordman, Tomas
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institute.
    Eriksson, L. C.
    Olsson, J. M.
    Reduction of ubiquinone by lipoamide dehydrogenase: An antioxidant regenerating pathway2001In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 5, p. 1486-1490Article in journal (Refereed)
    Abstract [en]

    Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min-1 in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mM and was even further increased at 0.5 mM. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP+ ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.

  • 755.
    Xia, Ling
    et al.
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Nordman, Tomas
    Södertörn University, Avdelning Naturvetenskap. Karolinska Institutet.
    Olsson, J M
    Damdimopoulos, A
    Björkhem-Bergman, L
    Nalvarte, I
    Eriksson, L C
    Arner, E S J
    Spyrou, Giannis
    Södertörn University, Avdelning Naturvetenskap. Karolinska Instiutet.
    Björnstedt, Mikael
    Karolinska Institutet.
    The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone - A novel mechanism for defense against oxidative stress2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 4, p. 2141-2146Article in journal (Refereed)
  • 756.
    Xue, Yongtao
    et al.
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Haas, S A
    Max-Plank Institute for Molecular Genetics, Berlin, Germany.
    Brino, L
    Eurogentec SA, Seraing, Belgium.
    Gusnanto, A
    Karolinska Institute.
    Reimers, M
    Karolinska Institute.
    Talibi, D
    Eurogentec SA, Seraing, Belgium.
    Vingron, M
    Max-Plank Institute for Molecular Genetics, Berlin, Germany.
    Ekwall, Karl
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    Wright, Anthony P H
    Södertörn University, School of Chemistry, Biology, Geography and Environmental Science. Karolinska Institute.
    A DNA microarray for fission yeast: minimal changes in global gene expression after temperature shift2004In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 21, no 1, p. 25-39Article in journal (Refereed)
    Abstract [en]

    Completion of the fission yeast genome sequence has opened up possibilities for post-genomic approaches. We have constructed a DNA microarray for genome-wide gene expression analysis in fission yeast. The microarray contains DNA fragments, PCR-amplified from a genomic DNA template, that represent >99% of the 5000 or so annotated fission yeast genes, as well as a number of control sequences. The GenomePRIDE software used attempts to design similarly sized DNA fragments corresponding to gene regions within single exons, near the 3'-end of genes that lack homology to other fission yeast genes. To validate the design and utility of the array, we studied expression changes after a 2 h temperature shift from 25degreesC to 36degreesC, conditions widely used when studying temperature-sensitive mutants. Obligingly, the vast majority of genes do not change more than two-fold, supporting the widely held view that temperature-shift experiments specifically reveal phenotypes associated with temperature-sensitive mutants. However, we did identify a small group of genes that showed a reproducible change in expression. Importantly, most of these corresponded to previously characterized heat-shock genes, whose expression has been reported to change after more extreme temperature shifts than those used here.. We conclude that the DNA microarray represents a useful resource for fission yeast researchers as well as the broader yeast community, since it will facilitate comparison with the distantly related budding yeast, Saccharomyces cerevisiae. To maximize the utility of this resource, the array and its component parts are fully described in On-line Supplementary Information and are also available commercially.

  • 757.
    Xue-Franzen, Yongtao
    Södertörn University, School of Life Sciences, Molecular biology. Karolinska Intitutet.
    DNA microarray approaches to understanding the regulation and evolution of gene expression networks2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA microarray technology allows biological and medical research to shift from investigation of individual functions of a few related genes to the whole genome level. This creates opportunities for discovery of complex and coordinated transcriptional networks in biological systems. The aim of this thesis has been to study gene regulation and evolution using yeast responses to environmental cues as a model system. We first developed and validated a fission yeast cDNA microarray for genome-wide expression analysis (Paper I). It is the first commercially available fission yeast microarray, which presents a useful resouce for yeast researchers and provides information required to contruct the array from scratch. Next, we characterised the gene regulatory networks involved in the pheromone response (Paper II) and investigate the role of Gcn5 transcription co-regulator, a histone acetyltransferase (HAT), in re-programming gene expression during the salt stress response in fission yeast (Paper III). We further investigated evolutionary conservation and divergence of Gcn5 in gene regulation by comparing its role in the evolutionarily distantly related yeast species. The parallel study of the fission yeast and budding yeast showed that Gcn5 has a conserved physiological role in salt stress responses, but it regulates diverged sets of stress response genes potentially via distinct mechanisms (paper IV). Finally, we investigated interactions between different HATs and between HATs and HDACs (histone deacetylases). Phenotypic studies and gene expression profiling revealed that Gcn5 has overlapping functions with another HAT, Mst2, in the stress response and DNA damage repair (Paper V). We found that the HDAC Clr3 acts antagonistically to Gcn5 in transcriptional elongation and stress responses (Paper VI).

  • 758.
    Xue-Franzen, Yongtao
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Kjaerulff, Soren
    University of Copenhagen, Copenhagen, Denmark.
    Holmberg, Christian
    University of Copenhagen, Copenhagen, Denmark.
    Wright, Anthony
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Nielsen, Olaf
    University of Copenhagen, Copenhagen, Denmark.
    Genomewide identification of pheromone-targeted transcription in fission yeast2006In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, p. 303-, article id 303Article in journal (Refereed)
    Abstract [en]

    Background: Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which then enter meiosis and sporulate. Prior to mating, M and P cells communicate with diffusible mating pheromones that activate a signal transduction pathway in the opposite cell type. The pheromone signalling orchestrates mating and is also required for entry into meiosis. Results: Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone signalling independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. Gene disruption experiments demonstrated the involvement of newly discovered pheromone-induced genes in the differentiation process. We have mapped Gene Ontology ( GO) categories specifically associated with pheromone induction. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M- specific genes and one new P-specific gene. Conclusion: We found that the pheromone response was very similar in M and P cells. Surprisingly, pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. Our results suggest that the SteII transcription factor is responsible for the majority of pheromone-induced transcription. Finally, most cell-type specific genes now appear to be identified in fission yeast.

  • 759.
    Yakusheva, Natalya
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies, Environmental Science.
    Socio-demographic changes in and around protected areas and management responses: Case studies from the Carpathians2016In: Parks of the Future:: Protected Areas in Europe Challenging Regional and Global Change / [ed] Thomas Hammer, Ingo Mose, Dominik Siegrist, Norbert Weixlbaumer, Munich: oekom verlag, 2016, p. 225-240Chapter in book (Refereed)
  • 760. Yang, Y
    et al.
    Griffiths, W J
    Nordling, M
    Nygren, Jonas
    Södertörn University, Avdelning Naturvetenskap. Karoliska Institute.
    Möller, L
    Bergman, Jan
    Liepinsh, E
    Otting, G
    Gustafsson, J A
    Rafter, J
    Sjövall, J
    Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites2000In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, no 50, p. 15585-15591Article in journal (Refereed)
    Abstract [en]

    The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given C-14-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.

  • 761.
    Yildirim, Håkan H.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Structural diversity of the lipid A and core oligosaccharide moieties of the lipopolysaccharides from nontypeable and serotype f Haemophilus influenzae2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis describes structural studies of the oligosaccharide and lipid A moieties of lipopolysaccharides (LPSs) isolated from disease-causing Haemophilus influenzae strains. The nontypeable strains were clinical isolates from the middle ear of children suffering from otitis media and the serotype f strains had been collected from three adults with respiratory tract infections. The LPS molecules are situated on the cell wall of H. influenzae strains and they play a very important role in colonization, infection, evasion of host immune system and inflammatory response. Previous studies have implicated the heterogeneous repertoire of LPS structures within a strain and mimicry of human cell wall structures to be involved in the diseasecausing behavior of this organism. Structural analysis of the oligosaccharide moieties with advanced applications of nuclear magnetic resonance (NMR) and various electrospray ionization mass spectrometry (ESI-MS) techniques revealed novel structural features in each of the investigated strains. All of the strains displayed a very complex mixture of LPS structures that differed between and within the pathogens. Moreover, all of the strains had the capacity to express mimics of human glycolipids. The genetic basis for LPS biosynthesis for H. influenzae is established for the strain of which the complete genome has been determined. In this thesis the function of the genes involved in the biosynthesis of LPS was investigated in a nontypeable strain by using the combination of genetic engineering and structural analysis. The synergy of genomics and analytical carbohydrate chemistry led to the identification of novel structural epitopes, and furthermore, enabled us to identify a new function for one of these genes. The most recent structural study of lipid A from H. influenzae was conducted in 1988 on a mutant strain. The results of that study established the presence of only one lipid A structure. in this thesis we investigated lipid A from both nontypeable and serotype wild type strains by performing tandem ESI-MS and the results confirmed earlier findings but also evidenced other lipid A structures previously not associated with H. influenzae. Moreover, all of the strains exhibited a heterogeneous population of lipid A molecules.

  • 762.
    Yildirim, Håkan H.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Hood, D W
    University of Oxford, John Radcliffe Hostpital, Oxford, UK.
    Moxon, E R
    University of Oxford, John Radcliffe Hostpital, Oxford, UK.
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Structural analysis of lipopolysaccharides from Haemophilus influenzae serotype f - Structural diversity observed in three strains2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 15, p. 3153-3167Article in journal (Refereed)
    Abstract [en]

    Structural elucidation of the lipopolysaccharide (LPS) from three serotype f Haemophilus influenzae clinical isolates RM6255, RM7290 and RM6252 has been achieved using NMR spectroscopy techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. This is the first study to report structural details on LPS from serotype f strains. We found that the LPSs of all strains were highly heterogeneous mixtures of glycoforms expressing the common H. influenzae structural element l-alpha-d-Hepp -(1-->2)-[P Etn-->6]-l-alpha-d-Hepp -(1-->3)-[beta-d-Glcp -(1-->4)]-l-alpha-d-Hepp -(1-->5)-[PP Etn-->4]-alpha-Kdo-(2-->6)-lipid A with variable length of OS chains linked to each of the heptoses. The terminal heptose (HepIII) in RM6255 is substituted at the O-3 position by a beta-d-Glcp residue whereas HepIII in strains RM7290 and RM6252 is substituted at O-2 by the globoside unit (alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glc) or truncated versions thereof. The central heptose (HepII) is substituted by an alpha-d-Galp -(1-->4)-beta-d-Galp -(1-->4)-beta-d-Glcp -(1-->4)-alpha-d-Glcp unit in RM7290 and RM6252 or truncated versions thereof. Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elongating from HepII (beta-d-Glcp -(1-->4)-alpha-d-Glcp ). ESI-MSn on dephosphorylated and permethylated OS provided information on the existence of additional minor isomeric glycoforms.

  • 763.
    Yildirim, Håkan H.
    et al.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Li, J J
    National Research Council of Canada, Ottawa, Canada.
    Richards, J C
    National Research Council of Canada, Ottawa, Canada.
    Hood, D W
    John Radcliffe Hospital, Oxford, UK.
    Moxon, E R
    John Radcliffe Hospital, Oxford, UK.
    Schweda, Elke K. H.
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    An alternate pattern for globoside oligosaccharide expression in Haemophilus influenzae lipopolysaccharide: Structural diversity in nontypeable strain 11242005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 13, p. 5207-5224Article in journal (Refereed)
    Abstract [en]

    Common structural motifs of Haemophilus influenzae lipopolysaccharide (LPS) are globotetraose [beta-D-GalpNAc-(1 -> 3)-alpha-D-Galp-(1 -> 4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp] and its truncated versions globoside [alpha-D-Galp-(1 -> 4)-beta-D-Galp-(1 -> 4)-beta-D-Glcp] and lactose [beta-D-Galp-(1 -> 4)-beta-D-Glcp] linked to the tenninal heptose (HepIII) of the triheptosyl inner-core moiety L-alpha-D-Hepp-(1 -> 2)-[PEA -> 6]-L-alpha-D-Hepp-(1 -> 3)L-alpha-D-Hepp-(1 -> 5)-[PPEA -> 4]-alpha-Kdo-(2 -> 6)-lipid A. We report here structural studies of LPS from nontypeable H. influenzae strain 1124 expressing these motifs linked to both the proximal heptose (HepI) and HepIII at the same time. This novel finding was obtained by structural studies of LPS using NMR techniques and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS) as well as ESI-MSn on permethylated dephosphorylated OS. The use of defined mutants allowed us to confirm structures unambiguously and understand better the biosynthesis of each of the globotetraose units. We found that lgtC is involved in the expression of beta-D-Galp-(1 -> 4)-beta-D-Galp in both extensions, whereas lic2A directs only the expression Of beta-D-Ga1p-(1 -> 4)-beta-D-Glcp when linked to HepIII. The LPS of NTHi strain 1124 contained sialylated glycoforms that were identified by CE-ESI-MS/MS. A common sialylated structure in H. influenzae LPS is sialyllactose linked to HepIII. This structure exists in strain 1124. However, results for the lpsA mutant indicate that sialyllactose extends from HepI as well, a molecular environment for sialyllactose in H. influenzae that has not been reported previously. In addition, the LPS was found to carry phosphoryleholine, O-linked glycine, and a third PEA group which was linked to O3 of HepIII.

  • 764.
    Yildirim, Håkan H
    et al.
    Södertörn University, School of Life Sciences. Karolinska institutet.
    Li, J J
    National Research Council, Ottawa, Canada.
    Richards, J C
    National Research Council, Ottawa, Canada.
    Hood, D W
    John Radcliffe Hospital, Oxford, UK.
    Moxon, E R
    John Radcliffe Hospital, Oxford, UK.
    Schweda, Elke K H
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation2005In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 340, no 17, p. 2598-2611Article in journal (Refereed)
    Abstract [en]

    The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, L-alpha-D-Hepp-(1 -> 2)-[PEtn -> 6]-L-alpha-D-Hepp-(1 -> 3)-[beta-D-Glcp-(1 -> 4)]-L-alpha-D-Hepp-(1 -> 5)-[PPEtn -> 4]-alpha-Kdo-(2 -> 6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEW at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1 -> 4)-beta-D-Glcp-(1 -> or beta-D-Glcp-(1 ->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MS" in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS.

  • 765.
    Youssef, Ninwa
    Södertörn University, School of Natural Sciences, Technology and Environmental Studies.
    Analysis of conserved microRNA targets in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster.2013Independent thesis Basic level (degree of Bachelor), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    MicroRNA (miRNA) is small regulatory non-coding single stranded RNA molecule that can repress protein expression either at transcriptional or translational level. Since their discoveries in nematodes in the early 1990´s extensive research have shown that this mechanism is conserved across species. Because the miRNA is so small, about 22 nucleotides (nt) long and only requires a minimum of 6nt to interact imperfect with its intended target 3´UTR, therefore a single miRNA could potentially have hundreds of potential targets, which have been suggested by computational prediction.

    The goal of the project is to experimentally verify three predicted Caenorhabditis elegans mir-2 miRNA­ targets in cell culture, with as candidate targets fos-1, mek-1 and sel-5.  In addition C. elegans mir-2 and its mechanism is conserved in Drosophila Melanogaster, miR-2. We want to elucidate if not only mir-2 miRNA is cross species conserved but also it targets. To test this hypothesis we selected the following predicted mir-2 target candidate genes: C. Elegans iff-1 and Drosophila Melanogaster protein ortholog eIF-5A.

    Validation of miRNA and its functionality was done by transfecting cells with a luciferase-3´UTR reporter only or luciferase-3´UTR and a miRNA-expression plasmid. After the reporter gene was induced, cells were harvested and the luciferase activity measured and the results normalized and compared. Unfortunately our data were inconclusive and future experiments are needed to give a clear picture.

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  • 766. Zhang, H Q
    et al.
    Li, Z L
    Viklund, E K
    Strömblad, Staffan
    Södertörn University, Avdelning Naturvetenskap.
    P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration2002In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 158, no 7, p. 1287-1297Article in journal (Refereed)
    Abstract [en]

    P21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. Science. 283: 2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alphavbeta5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alphavbeta5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alphavbeta5. Functionally, PAK4 induced integrin alphavbeta5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alphavbeta5 and selectively promotes integrin alphavbeta5-mediated cell migration.

  • 767. Zhang, X F
    et al.
    Meining, Winfried
    Södertörn University, Avdelning Naturvetenskap.
    Cushman, M
    Haase, I
    Fischer, M
    Bacher, A
    Ladenstein, R
    A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus2003In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 328, no 1, p. 167-182Article in journal (Refereed)
    Abstract [en]

    6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear. Here, we report four crystal structures of the enzyme from the hyperthermophilic bacterium Aquifex aeolicus in complex with different inhibitor compounds. The structures were refined at resolutions of 1.72 Angstrom, 1.85 Angstrom, 2.05 Angstrom and 2.2 Angstrom, respectively. The inhibitors have been designed in order to mimic the substrate, the putative reaction intermediates and the final product. Structural comparisons of the native enzyme and the inhibitor complexes as well as the kinetic data of singlesite mutants of lumazine synthase from Bacillus subtilis showed that several highly conserved residues at the active site, namely Phe22, His88, Arg127, Lys135 and Glu138 are most likely involved in catalysis. A structural model of the catalytic process, which illustrates binding of substrates, enantiomer specificity, proton abstraction/donation, inorganic phosphate elimination, formation of the Schiff base and cyclization is proposed.

  • 768. Zhang, X F
    et al.
    Meining, Winfried
    Södertörn University, Avdelning Naturvetenskap.
    Fischer, M
    Bacher, A
    Ladenstein, R
    X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 angstrom resolution: Determinants of thermostability revealed from structural comparisons2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 306, no 5, p. 1099-1114Article in journal (Refereed)
    Abstract [en]

    An open reading frame optimized for expression of 6,7-dimethyl-8-ribityllumazine synthase of the hyperthermophilic bacterium Aquifex aeolicus in Escherichia coli was synthesized and expressed in a recombinant E. coli strain to a level of around 15%. The recombinant protein was purified by heat-treatment and gel-filtration. The protein was crystallized in the cubic space group 123 with the cell dimensions a = b = c = 180.8 Angstrom, and diffraction data were collected to 1.6 Angstrom resolution. The structure was solved by molecular replacement using lumazine synthase from Bacillus subtilis as search model. The structure of the A. aeolicus enzyme was refined to a resolution of 1.6 Angstrom. The spherical protein consists of 60 identical subunits with strict icosahedral 532 symmetry. The subunit fold is closely related to that of the B. subtilis enzyme (rmsd 0.80 Angstrom). The extremely thermostable lumazine synthase from A. aeolicus has a melting temperature of 119.9 degreesC. Compared to other icosahedral and pentameric lumazine synthases, the A. aeolicus enzyme has the largest accessible surface presented by charged residues and the smallest surface presented by hydrophobic residues. It also has the largest number of ion-pairs per subunit. Two ion-pair networks involving two, respectively three, stacking arginine residues assume a distinct role in linking adjacent subunits. The findings indicate the influence of the optimization of hydrophobic and ionic contacts in gaining thermostability.

  • 769.
    Zhu, Xuefeng
    et al.
    Karolinska Institutet.
    Wirén, Marianna
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Sinha, Indranil
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Rasmussen, Nina N
    Institute of Molecular Biology, Copenhagen, Denmark.
    Linder, Tomas
    Karolinska Institutet.
    Holmberg, Steen
    Institute of Molecular Biology, Copenhagen, Denmark.
    Ekwall, Karl
    Södertörn University, School of Life Sciences. Karolinska Institutet.
    Gustafsson, Claes M
    Karolinska Institutet.
    Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions2006In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 22, no 2, p. 169-178Article in journal (Refereed)
    Abstract [en]

    Mediator exists in a free form containing the Med12, Med13, CDK8, and CycC subunits (the Srb8-11 module) and a smaller form, which lacks these four subunits and associates with RNA polymerase II (Pol II), forming a holoenzyme. We use chromatin immunoprecipitation (ChIP) and DNA microarrays to investigate genome-wide localization of Mediator and the Srb8-11 module in fission yeast. Mediator and the Srb8-11 module display similar binding patterns, and interactions with promoters and upstream activating sequences correlate with increased transcription activity. Unexpectedly, Mediator also interacts with the downstream coding region of many genes. These interactions display a negative bias for positions closer to the 5' ends of open reading frames (ORFs) and appear functionally important, because downregulation of transcription in a temperature-sensitive med17 mutant strain correlates with increased Mediator occupancy in the coding region. We propose that Mediator coordinates transcription initiation with transcriptional events in the coding region of eukaryotic genes.

  • 770. Åslund, F.
    et al.
    Berndt, Kurt D
    Karolinska Institutet.
    Holmgren, A.
    Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria1997In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 272, no 49, p. 30780-30786Article in journal (Refereed)
    Abstract [en]

    Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX1X2C). Standard state redox potential (E degrees ') values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E degrees ' resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E degrees ' values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E degrees ' = -233 and -198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E degrees ' values from a number of proteins ranging from -270 mV for E. coli Trx to -124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as -240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

  • 771. Åslund, F
    et al.
    Nordstrand, K
    Berndt, Kurt D
    Karolinska Institutet.
    Nikkola, M
    Bergman, T
    Ponstingl, H
    Jornvall, H
    Otting, G
    Holmgren, A
    Glutaredoxin-3 from Escherichia coli: Amino acid sequence, H-1 and N-15 NMR assignments, and structural analysis1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 12, p. 6736-6745Article in journal (Refereed)
    Abstract [en]

    The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. in addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys(65)) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific H-1 and N-15 nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, (3)J(HN alpha) coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low K-cat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grxl display large functional differences in in vitro protein disulfide oxide-reduction reactions.

13141516 751 - 771 of 771
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