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  • 151.
    Domkin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins2014In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, p. 5539-Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the formation of 2'-deoxyribonucleotides. Each polypeptide of the large subunit of eukaryotic RNRs contains two redox-active cysteine pairs, one in the active site and the other at the C-terminus. In each catalytic cycle, the active-site disulfide is reduced by the C-terminal cysteine pair, which in turn is reduced by thioredoxins or glutaredoxins. Dithiols such as DTT are used in RNR studies instead of the thioredoxin or glutaredoxin systems. DTT can directly reduce the disulfide in the active site and does not require the C-terminal cysteines for RNR activity. Here we demonstrate that the phosphines tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THP) are efficient non-thiol RNR reductants, but in contrast to the dithiols DTT, bis(2-mercaptoethyl)sulfone (BMS), and (S)-(1,4-dithiobutyl)-2-amine (DTBA) they act specifically via the C-terminal disulfide in a manner similar to thioredoxin and glutaredoxin. The simultaneous use of phosphines and dithiols results in ~3-fold higher activity compared to what is achieved when either type of reductant is used alone. This surprising effect can be explained by the concerted action of dithiols on the active-site cysteines and phosphines on the C-terminal cysteines. As non-thiol and non-protein reductants, phosphines can be used to differentiate between the redox-active cysteine pairs in RNRs.

  • 152.
    Domkin, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 21, p. 18574-8Article in journal (Refereed)
    Abstract [en]

    The ribonucleotide reductase system in Saccharomyces cerevisiae includes four genes (RNR1 and RNR3 encoding the large subunit and RNR2 and RNR4 encoding the small subunit). RNR3 expression, nearly undetectable during normal growth, is strongly induced by DNA damage. Yet an rnr3 null mutant has no obvious phenotype even under DNA damaging conditions, and the contribution of RNR3 to ribonucleotide reduction is not clear. To investigate the role of RNR3 we expressed and characterized the Rnr3 protein. The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. However, a strong synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated in experiments with the catalytically inactive Rnr1-C428A mutant, which increased the endogenous activity of Rnr3 by at least 10-fold. In vivo, the levels of Rnr3 after DNA damage never reached more than one-tenth of the Rnr1 levels. We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme, which may be important when Rnr1 is limiting for dNTP production. In complex with inactive Rnr1-C428A, the activity of Rnr3 is controlled by effector binding to Rnr1-C428A. This result indicates cross-talk between the Rnr1 and Rnr3 polypeptides of the large subunit.

  • 153. Drotz, Stina Harrysson
    et al.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nilsson, Mats B
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Öquist, Mats G
    Both catabolic and anabolic heterotrophic microbial activity proceed in frozen soils2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 49, p. 21046-21051Article in journal (Refereed)
    Abstract [en]

    A large proportion of the global soil carbon pool is stored in soils of high-latitude ecosystems in which microbial processes and production of greenhouse gases proceed during the winter months. It has been suggested that microorganisms have limited ability to sequester substrates at temperatures around and below 0 °C and that a metabolic shift to dominance of catabolic processes occurs around these temperatures. However, there are contrary indications that anabolic processes can proceed, because microbial growth has been observed at far lower temperatures. Therefore, we investigated the utilization of the microbial substrate under unfrozen and frozen conditions in a boreal forest soil across a temperature range from -9 °C to +9 °C, by using gas chromatography-isotopic ratio mass spectrometry and (13)C magic-angle spinning NMR spectroscopy to determine microbial turnover and incorporation of (13)C-labeled glucose. Our results conclusively demonstrate that the soil microorganisms maintain both catabolic (CO(2) production) and anabolic (biomass synthesis) processes under frozen conditions and that no significant differences in carbon allocation from [(13)C]glucose into [(13)C]CO(2) and cell organic (13)C-compounds occurred between +9 °C and -4 °C. The only significant metabolic changes detected were increased fluidity of the cell membranes synthesized at frozen conditions and increased production of glycerol in the frozen samples. The finding that the processes in frozen soil are similar to those in unfrozen soil has important implications for our general understanding and conceptualization of soil carbon dynamics in high-latitude ecosystems.

  • 154.
    Drotz, Stina Harrysson
    et al.
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences. Umeå.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences. Umeå.
    Öquist, Mats G
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences. Umeå.
    Effects of soil organic matter composition on unfrozen water content and heterotrophic CO2 production of frozen soils2010In: Geochimica et Cosmochimica Acta, ISSN 0016-7037, E-ISSN 1872-9533, Vol. 74, no 8, p. 2281-90Article in journal (Refereed)
    Abstract [en]

    Several recent studies have highlighted the importance of soil organic matter (SOM) mineralization at high latitudes during winter for ecosystem carbon (C) balances, and the ability of the soil to retain unfrozen water at sub-zero temperatures has been shown to be a major determinant of C mineralization rates. Further, SOM is believed to strongly influence the liquid water contents in frozen surface layers of boreal forest soils and tundra, but the mechanisms and specific factors involved are currently unknown. Here we evaluate the effects of the chemical composition of SUM on the amount of unfrozen water, the pore size equivalents in which unfrozen water can exist, and the microbial heterotrophic activity at sub-zero temperatures in boreal forest soils. To do this, we have characterized the chemical composition of SUM in forest soil samples (surface O-horizons) using solid state CP-MAS (cross polarization magic angle spinning) NMR spectroscopy. The acquired information was then used to elucidate the extent to which different fractions of SUM can explain the observed variations in unfrozen water content, pore size equivalents, and biogenic CO2 production rates in the examined soil samples under frozen conditions (-4 degrees C). The data evaluation was done by the use of principal component analysis (PCA) and projections to latent structures by means of partial least square (PLS). We conclude that aromatic, O-aromatic, methoxy/N-alkyl and alkyl C are the major SOM components affecting frozen boreal forest soil's ability to retain unfrozen water and sustain heterotrophic activity (95% confidence level). Our results reveal that solid carbohydrates have a significant negative impact (95% confidence level) on CO2 production in frozen boreal spruce forest soils, in contrast to the positive effects of carbohydrate polymers during unfrozen conditions. We conclude that the hierarchy of environmental factors controlling SOM mineralization changes as soils freeze. The effect of SUM composition on pore size distribution and unfrozen water content has a superior influence on SUM mineralization and hence on heterotrophic CO2 production of frozen soils. (C) 2010 Elsevier Ltd. All rights reserved.

  • 155.
    Dubbaka Venu, Pradeep Reddy
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Molecular studies of intra-oocyte phosphatidylinositol 3 kinase (PI3K) signaling pathway in controlling female fertility2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The primordial follicle pool is the main source of developing follicles in the ovary. The length of reproductive life and the onset of menopause are governed by the amount of primordial follicles in the ovary. The genetic factors and molecular mechanisms that maintain the primordial follicles in a dormant and surviving state for the whole of reproductive life are not well understood. The phosphatidylinositol 3 kinase (PI3K) signaling pathways in the oocyte that control oocyte growth and early follicular development are largely unknown. The major aim of this thesis was to investigate the functional role of the intra-oocyte PI3K pathway in the regulation of primordial follicle activation and survival.

     Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a major negative regulator of PI3K. The conditional deletion of Pten in the oocytes of primordial follicles led to the overgrowth of oocytes and activation of the entire pool of primordial follicles. There were higher numbers of activated primordial follicles at postnatal day 8 (PD8) in ovaries lacking PTEN in oocytes; by PD35 all the primordial follicles were activated and all the follicles were depleted by 12 weeks, causing premature ovarian failure (POF). In addition, the rate of follicular death that occurs during sexual maturity is reduced in ovaries that lack PTEN in oocytes. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling and upregulation of both expression and activation of ribosomal protein S6 (rpS6). The overactivation of primordial follicles in ovaries that lack PTEN in oocytes is believed to be due to elevated expression and activation of rpS6. PTEN in oocytes is indispensable for the maintenance of primordial follicles in dormancy.

     To study the role of the intra-oocyte PI3K signaling pathway in controlling the survival and maintenance of primordial follicles, 3-phosphoinositide-dependent protein kinase-1 (PDK1) was deleted in oocytes of primordial follicle. The loss of Pdk1 in oocytes led to the depletion of most primordial follicles around the onset of sexual maturity, causing POF during early adulthood. Furthermore, the activation of Akt, p70 S6 kinase 1 (S6K1), and rpS6 was impaired in oocytes that lacked PDK1. The suppressed PDK1–Akt–S6K1–rpS6 signaling in oocytes appears to be responsible for the loss of primordial follicles. The excessive activation of primordial follicles seen in the absence of Pten in oocytes could be reversed by concurrent deletion of Pdk1. In addition, the elevated activation of Akt and S6K1 in the absence of PTEN in oocytes was not observed in PTEN and PDK1 double mutant mice. Similarly, the hyperphosphorylation of rpS6 in oocytes that lack PTEN was prevented in double mutant mice, which was most likely due to downregulation of S6K1 activation. Thus, inactivation of rpS6 in double mutant mice might be the reason for the prevention of excessive primordial follicular activation and survival.

     PTEN and PDK1 in oocytes are essential for the maintenance of quiescence and survival of primordial follicles. The molecular network involving PI3K/PTEN–PDK1 signaling in oocyte controls the survival, loss, and activation of primordial follicles, which together govern reproductive aging and determine the length of reproductive life in females. The results of the above studies indicate that the mammalian oocyte serves as the seat of programming of follicular activation and survival.

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  • 156.
    Dubbaka Venu, Pradeep Reddy
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Adhikari, Deepak
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zheng, Wenjing
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liang, Shawn
    Hämäläinen, Tuula
    Tohonen, Virpi
    Ogawa, Wataru
    Noda, Tetsuo
    Volarevic, Sinisa
    Huhtaniemi, Ilpo
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    PDK1 signaling in oocytes controls reproductive aging and lifespan by manipulating the survival of primordial follicles2009In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 18, no 15, p. 2813-2824Article in journal (Refereed)
    Abstract [en]

    The molecular mechanisms that control reproductive aging and menopausal age in females are poorly understood. Here, we provide genetic evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) signaling in oocytes preserves reproductive lifespan by maintaining the survival of ovarian primordial follicles. In mice lacking the PDK1-encoding gene Pdk1 in oocytes, the majority of primordial follicles are depleted around the onset of sexual maturity, causing premature ovarian failure (POF) during early adulthood. We further showed that suppressed PDK1-Akt-p70 S6 kinase 1 (S6K1)-ribosomal protein S6 (rpS6) signaling in oocytes appears to be responsible for the loss of primordial follicles, and mice lacking the Rps6 gene in oocytes show POF similar to that in Pdk1-deficient mice. In combination with our earlier finding that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in oocytes suppresses follicular activation, we have now pinpointed the molecular network involving phosphatidylinositol 3 kinase (PI3K)/PTEN-PDK1 signaling in oocytes that controls the survival, loss and activation of primordial follicles, which together determine reproductive aging and the length of reproductive life in females. Underactivation or overactivation of this signaling pathway in oocytes is shown to cause pathological conditions in the ovary, including POF and infertility.

  • 157.
    Dubbaka Venu, Pradeep Reddy
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liu, Lian
    Adhikari, Deepak
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jagarlamudi, Krishna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rajareddy, Singareddy
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yan
    Du, Chun
    Tang, Wenli
    Hämäläinen, Tuula
    Peng, Stanford L
    Lan, Zi-Jian
    Cooney, Austin J
    Huhtaniemi, Ilpo
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool2008In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 319, no 5863, p. 611-613Article in journal (Refereed)
  • 158. Dubois, Andre
    et al.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Helicobacter pylori is invasive and it may be a facultative intracellular organism.2007In: Cellular microbiology, ISSN 1462-5814, Vol. 9, no 5, p. 1108-16Article in journal (Refereed)
  • 159. Duchardt, Elke
    et al.
    Nilsson, Lennart
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study.2008In: Nucleic acids research, ISSN 1362-4962, Vol. 36, no 12, p. 4211-9Article in journal (Refereed)
    Abstract [en]

    Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.

  • 160. Dunny, Gary
    et al.
    Berntsson, Ronnie Per-Arne
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enterococcal sex pheromones: evolutionary pathways to complex, two-signal systems2016In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, no 11, p. 1556-1562Article, review/survey (Refereed)
    Abstract [en]

    Gram-positive bacteria carry out intercellular communication using secreted peptides. Important examples of this type of communication are the enterococcal sex pheromone systems, in which the transfer of conjugative plasmids is controlled by intercellular signaling among populations of donors and recipients. This review focuses on the pheromone response system of the conjugative plasmid pCF10. The peptide pheromones regulating pCF10 transfer act by modulating the ability of the PrgX transcription factor to repress the transcription of an operon encoding conjugation functions. Many Gram-positive bacteria regulate important processes, including the production of virulence factors, biofilm formation, sporulation, and genetic exchange using peptide-mediated signaling systems. The key master regulators of these systems comprise the RRNPP (RggRap/NprR/PlcR/PrgX) family of intracellular peptide receptors; these regulators show conserved structures. While many RRNPP systems include a core module of two linked genes encoding the regulatory protein and its cognate signaling peptide, the enterococcal sex pheromone plasmids have evolved to a complex system that also recognizes a second host-encoded signaling peptide. Additional regulatory genes not found in most RRNPP systems also modulate signal production and signal import in the enterococcal pheromone plasmids. This review summarizes several structural studies that cumulatively demonstrate that the ability of three pCF10 regulatory proteins to recognize the same 7-amino-acid pheromone peptide arose by convergent evolution of unrelated proteins from different families. We also focus on the selective pressures and structure/function constraints that have driven the evolution of pCF10 from a simple, single-peptide system resembling current RRNPPs in other bacteria to the current complex inducible plasmid transfer system.

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  • 161. Earp, Caroline
    et al.
    Rowbotham, Samuel
    Marjavaara, Lisette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cha, Rita S
    Acute sensitivity of DNA replication to reduction in dNTP pools following Mec1ATR inactivation2015In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, no Suppl. 1, p. S56-S56Article in journal (Other academic)
    Abstract [en]

    Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by a fatal mitotic catastrophe. The mec1 S phase arrest was proposed to stem from a defect in the Mec1-Rad53-Dun1 dependent removal of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), at the onset of S phase: According to this view, Sml1 removal and the ensuing RNR activation would promote the dNTP production necessary for genome duplication. In support for this view, dNTP levels in hypomorphic mec1 or rad53 mutants and a dun1∆ strain were shown to be reduced by as much as 46% compared to a MEC1 control strain. Notably however, nearly all analyses on a lethal mec1 allele (e.g. mec1∆ or mec1-kd [kinase dead]) have been performed in a strain background that was either deleted for SML1 or over-expressing RNR1, a requirement for maintaining viability of a mutant lacking Mec1's essential function. As a result, while it is clear that absence of Mec1 causes dNTP pool to decrease, the true extent of the reduction and whether it would be sufficient to account for the replication arrest remain elusive. Here, we addressed these questions utilizing a temperature sensitive mutant, mec1-4, which maintains its viability at permissive temperature in an otherwise wild-type background, circumventing the need to exogenously manipulate Sml1 and/or RNR activity. Results show that Mec1 inactivation leads to an S phase arrest and a ~17% reduction in dNTP pool; expression of a novel suppressor, GIS2 (glucose inhibition of gluconeogenic growth suppressor 2), rescues the arrest and partially restores the dNTP pool to ~ 93% of a control. Unexpectedly modest effects of mec1 and GIS2 on dNTP levels demonstrate that the arrest does result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to its availability

  • 162. Earp, Caroline
    et al.
    Rowbotham, Samuel
    Merényi, Gábor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Cha, Rita S
    S phase block following MEC1ATR inactivation occurs without severe dNTP depletion2015In: Biology open, ISSN 2046-6390, Vol. 4, no 12, p. 1739-1743Article in journal (Refereed)
    Abstract [en]

    Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.

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  • 163. Eberth, Alexander
    et al.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gremer, Lothar
    Dvorsky, Radovan
    Koessmeier, Katja T
    McMahon, Harvey T
    Ahmadian, Mohammad Reza
    A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 417, no 1, p. 371-377Article in journal (Refereed)
    Abstract [en]

    The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.

  • 164. Edlund, T
    et al.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rånby, M
    Hedén, L O
    Palm, G
    Holmgren, E
    Josephson, S
    Isolation of cDNA sequences coding for a part of human tissue plasminogen activator.1983In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 80, no 2, p. 349-52Article in journal (Refereed)
    Abstract [en]

    We have isolated a cDNA sequence coding for a part of human tissue plasminogen activator. mRNA coding for tissue plasminogen activator was partially purified, copied into double-stranded cDNA, and cloned into Escherichia coli. Two sets of partially overlapping oligodeoxynucleotide mixtures corresponding to all possible coding sequences for a known portion of the tissue plasminogen activator gene were prepared. One set was used as a probe to screen cDNA containing bacterial clones and both were used as probes in hybridization against purified plasmid DNA. Of 4,200 bacterial clones examined, 1 carried a plasmid that hybridized to both sets of oligonucleotides. This plasmid contained a 370-base-pair cDNA insert, which was shown by nucleotide sequence analysis to code for the cleavage site region in the one-chain form of the human tissue plasminogen activator.

  • 165.
    Ehlers, Ina
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    NMR studies of metabolites and xenobiotics: From time-points to long-term metabolic regulation2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science.

    The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs.

    Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches.

    The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects.

    The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.

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  • 166.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Augusti, Angela
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Betson, Tatiana R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B.
    Marshall, John D.
    Schleucher, Juergen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Detecting long-term metabolic shifts using isotopomers: CO2-driven suppression of photorespiration in C-3 plants over the 20th century2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 51, p. 15585-15590Article in journal (Refereed)
    Abstract [en]

    Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C-3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C-3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C-3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the similar to 100-ppm CO2 increase between similar to 1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.

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  • 167.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Augusti, Angela
    Betson, Tatiana R.
    Nilsson, Mats B.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Quantification of a metabolic shift towards photosynthesisin C3 plants driven by 20th-century CO2 riseManuscript (preprint) (Other academic)
    Abstract [en]

    Terrestrial vegetation currently absorbs approximately a third of the annual anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However,terrestrial net primary production is highly sensitive to atmospheric [CO2] and associated climatic changes. In C3-plants, which dominate terrestrial vegetation, netphotosynthesis depends on the ratio between gross photosynthesis and photorespiration, which strongly depends on [CO2]. However, our knowledge of feedbacks betweenterrestrial biomes and increasing atmospheric [CO2] is nearly entirely based on atmospheric inversion models and manipulation experiments, which do not reveal physiological mechanisms or are limited in duration and to step increases in [CO2]. By applying novel NMR (Nuclear Magnetic Resonance) spectroscopy methodology we examine isotopomer ratios of plant carbohydrates to probe shifts in the photosynthesis/photorespiration ratio in C3 plants over more than a century. Using herbarium samples of natural vascular plant species, crops and a Sphagnum species, we detect a consistent 35% increase in the 2photosynthesis/photorespiration ratio in responseto the ~100 ppm CO2 increase between approximately 1900 and 2013, with no evidencefor feedback regulation by the plants. Our data provide direct quantitative information on the “CO2 fertilization effect” over century time scales, thus addressing a major uncertainty in Earth system models, enabling improved predictions of the future [CO2] sink strength of terrestrial ecosystems. Further, relating the detected metabolic shift in crop plants to historic yield trends indicates that only a fraction of the increased net photosynthesis has translated into increased yield. Our results also demonstrate that archives of plant material contain metabolic information embedded in their isotopomer ratios covering centuries, bridging a fundamental gap between experimental plant science and paleoenvironmental studies.

  • 168. Ehlers, Ina
    et al.
    Augusti, Angela
    Köhler, Iris
    Wieloch, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zuidema, Pieter
    Robertson, Iain
    Nilsson, Mats
    Marshall, John
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Detecting plant-climate interactions over decades-millennia using NMR isotopomer analysis2016In: Geophysical Research Abstracts, 2016, Vol. 18, article id EGU2016-9141-2Conference paper (Refereed)
  • 169.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Betson, Tatiana R.
    Vetter, Walter
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Elucidating Turnover Pathways of Bioactive Small Molecules by Isotopomer Analysis: The Persistent Organic Pollutant DDT2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 10, p. e110648-Article in journal (Refereed)
    Abstract [en]

    The persistent organic pollutant DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane) is still indispensable in the fight againstmalaria, although DDT and related compounds  pose toxicological  hazards. Technical DDT contains the dichloro congenerDDD (1-chloro-4-[2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene)   as by-product, but  DDD is also formed by  reductive degradation of DDT in the environment. To differentiate between DDD formation pathways, we applied deuterium NMR spectroscopy to measure intramolecular deuterium distributions (2H isotopomer abundances) of DDT and DDD. DDD formed in the technical  DDT synthesis was strongly deuterium-enriched at one intramolecular position, which we traced back to 2H/1H fractionation of a chlorination step in the technical synthesis.  In contrast, DDD formed by reductive degradation was strongly depleted at the same position, which was due to the incorporation of 2H-depleted hydride equivalents during reductive degradation. Thus, intramolecular isotope distributions give mechanistic information on reaction pathways, and explain a puzzling difference in the whole-molecule 2H/1H ratio between DDT and DDD. In general, our results highlight that intramolecular isotope distributions are essential to interpret whole-molecule isotope ratios. Intramolecular isotope information allows distinguishing pathways of DDD formation, which is important to identify polluters or to assess  DDT turnover in the environment. Because  intramolecular isotope data directly reflect isotope fractionation of individual chemical reactions, they are broadly applicable to elucidate transformation pathways of smallbioactive molecules in chemistry, physiology and environmental science.

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  • 170.
    Ehlers, Ina
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Köhler, Iris
    Wieloch, Thomas
    Vlam, Mart
    van der Sleen, Peter
    Groenendijk, Peter
    Grabner, Michael
    Seim, Andrea
    Allen, Kathryn
    Wei, Liang
    Robertson, Iain
    Marshall, John
    Zuidema, Pieter A.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Limited suppression of photorespiration by 20th century atmospheric CO2 increase in trees worldwideManuscript (preprint) (Other academic)
    Abstract [en]

    Forests are a key component of the global carbon and hydrological cycle and forest responses to  environmental  drivers  create  important  feedbacks  to  these  cycles.  Photosynthetic efficiency of most forest tree species is strongly limited by photorespiration, a side reaction using O2 instead of CO2 as substrate, leading to a carbon loss for the plant. Photorespiration occurs in all trees and is reduced under elevated CO2 concentrations and increased under elevated temperature. Because the CO2 concentration of the atmosphere has increased in past decades, long-lived trees may have benefited from reduced photorespiration, but the temperature increase would have been a compensating detriment; but direct quantification of long-term changes in metabolic fluxes is lacking. Realistic forecasting of responses of trees and forests to future CO2 and temperature demands quantifying the reduction of photorespiration.  In  twelve  tree  species  from  five  continents,  we  observe  that photorespiration has been reduced by the CO2 increase during the past century, but for most the reduction is smaller than predicted from plant responses in CO2 alone. Comparison with data from a combined CO2 and temperature manipulation experiment shows that the reduced response can be explained by increases in leaf temperatures, which might result directly from increased  air  temperatures  or  indirectly  from  reduced  transpirative  cooling.  These  data suggest that global warming has already inhibited plant fertilization by increasing CO2, and that biomass increases may have been smaller than deduced from measurements of the heavy carbon isotope 13C. Observation of this centennial metabolic shift in tree physiology worldwide provides new insights into forest-climate feedbacks and can be used to improve coupled climate-vegetation models.

  • 171. Ehlers, Ina
    et al.
    Wieloch, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Groenendijk, Peter
    Vlam, Mart
    van der Sleen, Peter
    Zuidema, Pieter A.
    Robertson, Iain
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Enhanced photosynthetic efficiency in trees world-wide by rising atmospheric CO2 levels2014In: Geophysical Research Abstracts, 2014, Vol. 16, article id EGU2014-12587-1Conference paper (Refereed)
  • 172.
    Eklund, Lovsa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The effect of chemical treatment on the nanomechanical properties of Aβ-plaques in the brain tissues of patients with Alzheimer´s disease2016Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
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  • 173.
    Elfving, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Transcriptional regulation of mouse ribonucleotide reductase2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.

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  • 174.
    Elfving, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Thelander, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Characterization of the promoters of the mouse ribonucleotide reductase R1, R2 and p53R2-genesManuscript (preprint) (Other academic)
    Abstract [en]

    Reduction of ribonucleotides into deoxyribonucleotides is catalyzed by the enzyme ribonucleotide reductase. The mouse enzyme is composed of two protein subunits, the R1 protein and the R2/p53R2 protein, and its subunit constellation differs during the cell cycle. We report here how the promoters of each of these subunits are regulated during the cell cycle. Previous DNase footprinting experiments of the R1 and the R2 promoter gave us an idea of how these promoters are structured. The R1 promoter contains four elements; Inr, α (binding YY1), β (binding YY1) and γ, while the R2 promoter contains four different elements; TATA-box (binding TBP), CCAAT-box (binding NFY), E2F element (binding E2F4) and an upstream activating region. The p53R2 promoter is uncharacterized; only the transcription start has been suggested in Genebank.

    We found that activation of both subunits needed for S phase specific activity (R1 and R2) is dependent of release of the repressor E2F4 from each promoter. Previous results showed that the mouse R2 promoter harbors an E2F4 binding element and our result, using transient transfections, indicates that this is also the case for the mouse R1 promoter. Using primer extension experiments on the mouse p53R2 promoter we show that the transcription start colocalizes with an earlier unidentified Inr element similar to the Inr element in the mouse R1 promoter. Our transcription start site is localized 126 bp downstream from the start site indicated in Genebank. We also show that it is possible to partially purify the transcription factor(s) binding to the upstream activating region in the mouse R2 promoter by using phosphocellulose chromatography and oligonucleotides immobilized on magnetic beads.

  • 175.
    Elfving, Nils
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Functional studies of Mediator in Arabidopsis thaliana and Saccharomyces cerevisiae2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mediator has been shown to be essential for regulation of RNA Polymerase II mediated transcription. Mediator functions as an interface between the general transcriptional machinery and a multitude of DNA binding transcriptional regulators, although the molecular mechanism for the process is elusive. Mediator is a large complex of over twenty subunits, most of which are conserved from yeast to plants to mammals. Many of these subunits are essential for viability in yeast, and mutations in the corresponding genes have global effects on transcription. Mediator was originally identified in Saccharomyces cerevisiae, but has since been described in most eukaryotes. However, until recently the Mediator complex was not identified in plants. This thesis describes the first successful identification and isolation of the Mediator complex from the plant Arabidopsis thaliana. By raising antibodies against candidate A. thaliana Mediator subunits, we were able to purify a multisubunit protein complex. Mass spectrometry and bioinformatics analysis allowed us to identify 21 of these subunits as conserved Mediator components and six as A. thaliana specific subunits. Some of the genes that encode the identified Mediator subunits had earlier been described as components of specific regulatory pathways controlling for example cell proliferation and flowering time. Subsequent genetic analysis confirmed that the A. thaliana Mediator complex is important for several plant signaling pathways, including flowering and stress pathways. This thesis also describes identification of regulators that interact with the A. thaliana Mediator subunit Med25, previously identified as PFT1 (Phytochrome and Flowering Time 1) and implicated in regulation of flowering time in response to light quality. Finally, we describe the function of Mediator in S. cerevisiae using genome-wide approaches. We have carried out a transcriptional switch where half of the genome changes expression and determined Mediator occupancy across the genome before and after such a switch, using ChIP-SEQ on tagged subunits from different Mediator domains. Unexpectedly, we find that Mediator occupancy is limited at most promoters. However, at the highly occupied promoters, we see different modes of changes in occupancy as a result of the transcriptional switch. These highly occupied promoters control genes involved in different stress response pathways. Thus, our results suggest that Mediator function and composition differ considerably between different promoters.

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  • 176.
    Elfving, Nils
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chereji, Razvan
    Department of Physics and Astronomy and BioMaPS Institute for Quantitative Biology, Rutgers University.
    Larsson, Miriam
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozov, Alexandre
    Department of Physics and Astronomy and BioMaPS Institute for Quantitative Biology, Rutgers University.
    Broach, James
    Department of Molecular Biology, Princeton University.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mediator exists in multiple forms and is predominantly associated to promoters with low nuclesome densityManuscript (preprint) (Other academic)
  • 177.
    Elfving, Nils
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chereji, Razvan V.
    Bharatula, Vasudha
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozov, Alexandre V.
    Broach, James R.
    A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 9, p. 5468-5482Article in journal (Refereed)
    Abstract [en]

    The transcription factor Msn2 mediates a significant proportion of the environmental stress response, in which a common cohort of genes changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have applied genome-wide chromatin immunoprecipitation and nucleosome profiling to determine where Msn2 binds under stressful conditions and how that binding affects, and is affected by, nucleosome positioning. We concurrently determined the effect of Msn2 activity on gene expression following stress and demonstrated that Msn2 stimulates both activation and repression. We found that some genes responded to both intermittent and continuous Msn2 nuclear occupancy while others responded only to continuous occupancy. Finally, these studies document a dynamic interplay between nucleosomes and Msn2 such that nucleosomes can restrict access of Msn2 to its canonical binding sites while Msn2 can promote reposition, expulsion and recruitment of nucleosomes to alter gene expression. This interplay may allow the cell to discriminate between different types of stress signaling.

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  • 178.
    Elfving, Nils
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Davoine, Céline
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Benlloch, Reyes
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brännström, Kristoffer
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Müller, Dörte
    Nilsson, Anders
    Ulfstedt, Mikael
    Ronne, Hans
    Wingsle, Gunnar
    Nilsson, Ove
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The Arabidopsis thaliana Med25 mediator subunit integrates environmental cues to control plant development2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 20, p. 8245-8250Article in journal (Refereed)
    Abstract [en]

    Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

  • 179.
    Elluri, Sridhar
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
    Enow Oben Ayuk, Constance
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Vdovikova, Svitlana
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rompikuntal, Pramod K
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Dongre, Mitesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Carlsson, Sven
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pal, Amit
    Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 9, article id e106731Article in journal (Refereed)
    Abstract [en]

    Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.

    Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.

    Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.

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  • 180. Elmlund, Hans
    et al.
    Baraznenok, Vera
    Linder, Tomas
    Szilagyi, Zsolt
    Rofougaran, Reza
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hebert, Hans
    Lindahl, Martin
    Gustafsson, Claes M
    Cryo-EM reveals promoter DNA binding and conformational flexibility of the general transcription factor TFIID2009In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 17, no 11, p. 1442-1452Article in journal (Refereed)
    Abstract [en]

    The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the TFIID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.

  • 181.
    Eremenko, Ekaterina
    et al.
    Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Ben-Zvi, Anat
    National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Raveh, Dina
    Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
    Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, p. e58218-Article in journal (Refereed)
    Abstract [en]

    Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.

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  • 182.
    Erhagen, Björn
    et al.
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Öquist, Mats
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Haei, Mahsa
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Ilstedt, Ulrik
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Temperature response of litter and soil organic matter decomposition is determined by chemical composition of organic material2013In: Global Change Biology, ISSN 1354-1013, E-ISSN 1365-2486, Vol. 19, no 12, p. 3858-3871Article in journal (Refereed)
    Abstract [en]

    The global soil carbon pool is approximately three times larger than the contemporary atmospheric pool, therefore even minor changes to its integrity may have major implications for atmospheric CO2 concentrations. While theory predicts that the chemical composition of organic matter should constitute a master control on the temperature response of its decomposition, this relationship has not yet been fully demonstrated. We used laboratory incubations of forest soil organic matter (SOM) and fresh litter material together with NMR spectroscopy to make this connection between organic chemical composition and temperature sensitivity of decomposition. Temperature response of decomposition in both fresh litter and SOM was directly related to the chemical composition of the constituent organic matter, explaining 90% and 70% of the variance in Q10 in litter and SOM respectively. The Q10 of litter decreased with increasing proportions of aromatic and O-aromatic compounds, and increased with increased contents of alkyl- and O-alkyl carbons. In contrast, in SOM, decomposition was affected only by carbonyl compounds. To reveal why a certain group of organic chemical compounds affected the temperature sensitivity of organic matter decomposition in litter and SOM, a more detailed characterisation of the (13) C aromatic region using Heteronuclear Single Quantum Coherence (HSQC) was conducted. The results revealed considerable differences in the aromatic region between litter and SOM. This suggests that the correlation between chemical composition of organic matter and the temperature response of decomposition differed between litter and SOM. The temperature response of soil decomposition processes can thus be described by the chemical composition of its constituent organic matter, this paves the way for improved ecosystem modelling of biosphere feedbacks under a changing climate.

  • 183. Erickson, L A
    et al.
    Schleef, R R
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Loskutoff, D J
    The fibrinolytic system of the vascular wall.1985In: Clinics in haematology, ISSN 0308-2261, Vol. 14, no 2, p. 513-30Article in journal (Refereed)
    Abstract [en]

    The vascular endothelium produces both PAs and a PAI. The activities of these components in the circulation must be regulated precisely to ensure that normal vascular homeostasis is not compromised. The blood contains a number of molecules that may function in this way by either promoting or inhibiting the synthesis, release and/or activity of the PAs and PAI. It is clear that the regulation of this system is considerably more complex than previously thought. For example, the initiation of fibrin dissolution is influenced by a number of additional factors including fibrin itself, pro-activators, PAI, platelet components (including the PAI), and possibly by APC generated at the endothelial cell surface. Despite the many recent advances discussed above, little is known about the temporal control of the events leading to plasminogen activation during thrombus formation and dissolution. Obviously, such information must be obtained before more effective treatments of abnormal vascular fibrinolytic activity can be developed. In this chapter, we have described a number of reagents and assays that should aid in the quantification of the PAs and the PAI in plasma. Eventual utilization of these assays in a clinical setting may be valuable for the diagnosis and subsequent treatment of abnormalities of the vascular fibrinolytic system.

  • 184.
    Eriksson, Daniella
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Masterprogrammet i biomedicin.
    Drugs and Risk of Hip Fractures A descriptive study among hip fracture patients in Umeå, Sweden.2014Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
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  • 185.
    Eriksson, Per-Olof
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Li, Jinan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hellström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Spontaneous development of otitis media in plasminogen-deficient mice2006In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 296, no 7, p. 501-509Article in journal (Refereed)
    Abstract [en]

    Inflammatory conditions of the ear, otitis media, are one of the most common disease entities in children. In this study, the role of the plasminogen (plg)/plasmin system for the spontaneous development of chronic otitis media was investigated by the analysis of plg-deficient mice. Whereas essentially all of the wild-type control mice kept a healthy status of the middle ear, all the plg-deficient mice gradually developed chronic otitis media with various degrees of inflammatory changes during an 18-week observation period. Five bacterial strains were identified in materials obtained from the middle ear cavities of six plg-deficient mice. Morphological studies revealed the formation of an amorphous mass tissue and inflammatory changes in the middle ears of plg-deficient mice. Immunohistochemical studies further indicate a mass infiltration of neutrophils and macrophages as well as the presence of T and B cells in the middle ear mucosa of these mice. Extensive fibrin deposition and an abnormal keratin formation were also observed in the tympanic membrane, the middle ear cavity and external ear canal in these mice. These results suggest that plg plays an essential role in protecting against the spontaneous development of chronic otitis media. Our findings also suggest the possibility of using plg for clinical therapy of certain types of otitis media.

  • 186.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sheng, Nongfei
    Umeå University, Faculty of Medicine, Department of Odontology.
    Mårell, Lena
    Umeå University, Faculty of Medicine, Department of Odontology.
    Claesson, Rolf
    Umeå University, Faculty of Medicine, Department of Odontology.
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Strömberg, Nicklas
    Umeå University, Faculty of Medicine, Department of Odontology.
    Streptococcus Mutans Adhesin Biotypes that Match and Predict Individual Caries Development2017In: EBioMedicine, E-ISSN 2352-3964, Vol. 24, p. 205-215Article in journal (Refereed)
    Abstract [en]

    Dental caries, which affects billions of people, is a chronic infectious disease that involves Streptococcus mutans, which is nevertheless a poor predictor of individual caries development. We therefore investigated if adhesin types of S.mutans with sucrose-independent adhesion to host DMBT1 (i.e. SpaP A, B or C) and collagen (i.e. Cnm, Cbm) match and predict individual differences in caries development. The adhesin types were measured in whole saliva by qPCR in 452 12-year-old Swedish children and related to caries at baseline and prospectively at a 5-year follow-up. Strains isolated from the children were explored for genetic and phenotypic properties. The presence of SpaP B and Cnm subtypes coincided with increased 5-year caries increment, and their binding to DMBT1 and saliva correlated with individual caries scores. The SpaP B subtypes are enriched in amino acid substitutions that coincided with caries and binding and specify biotypes of S. mutans with increased acid tolerance. The findings reveal adhesin subtypes of S. mutans that match and predict individual differences in caries development and provide a rationale for individualized oral care.

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  • 187. Fa, M.
    et al.
    Bergström, F.
    Hägglöf, P.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wilczynska, M.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, L. B.-A.
    Ny, T.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites2000In: Structure, with Folding & Design, Vol. 8, no 4, p. 397-405Article in journal (Refereed)
  • 188. Fa, M
    et al.
    Bergström, F
    Karolin, J
    Johansson, L B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 12, p. 3729-34Article in journal (Refereed)
    Abstract [en]

    The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

  • 189. Fa, M
    et al.
    Karolin, J
    Aleshkov, S
    Strandberg, L
    Johansson, L B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.1995In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 34, no 42, p. 13833-40Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

  • 190.
    Fa, Ming
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites2000In: Structure, Vol. 8, no 4, p. 397-405Article in journal (Refereed)
    Abstract [en]

    Background: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin–protease complex have been proposed. In the absence of a spatial structure of a serpin–protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial.

    Results: We used a recently developed method for making precise distance measurements, based on donor–donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1′) were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into β sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease–inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker.

    Conclusions: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

  • 191.
    Fa, Ming
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy: Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms2000In: European Journal of Biochemistry, Vol. 267, no 12, p. 3729-34Article in journal (Refereed)
    Abstract [en]

    The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY® probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57 ± 4 Å and 63 ± 3 Å, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into β-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

  • 192. Fagerberg, David
    et al.
    Angström, Jonas
    Halim, Adnan
    Hultberg, Anna
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hammarström, Lennart
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Teneberg, Susann
    Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human alpha-1,3/4-fucosyltransferase in FVB/N mouse stomach.2009In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 19, no 2, p. 182-191Article in journal (Refereed)
    Abstract [en]

    The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.

  • 193. Falahati, Hanieh
    et al.
    Pazhang, Mohammad
    Zareian, Shekufeh
    Ghaemi, Nasser
    Rofougaran, Reza
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rezaie, Alireza R
    Khajeh, Khosro
    Transmitting the allosteric signal in methylglyoxal synthase2013In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 26, no 7, p. 445-452Article in journal (Refereed)
    Abstract [en]

    The homohexameric enzyme methylglyoxal synthase (MGS) converts dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate. This enzyme is allosterically inhibited by phosphate. The allosteric signal induced by phosphate in MGS from Thermus sp. GH5 (TMGS) has been tracked by site-directed mutagenesis, from the binding site of phosphate to the pathways that transmit the signal, and finally to the active site which is the receiver of the signal. In TMGS, Ser-55 distinguishes the inhibitory phosphate from the phosphoryl group of the substrate, DHAP, and transmits the allosteric signal through Pro-82, Arg-97 and Val-101 to the active site. Furthermore, the addition of a C-terminal tail to TMGS reinforces the allosteric signal by introducing a new salt bridge between Asp-10 and an Arg in this tail. Lastly, the active site amino acid, Gly-56, is shown to be involved in both allostery and phosphate elimination step from DHAP by TMGS. Interestingly, some of the mutations also trigger homotropic allostery, supporting the hypothesis that allostery is an intrinsic property of all dynamic proteins. The details of the TMGS allosteric network discussed in this study can serve as a model system for understanding the enigmatic allosteric mechanism of other proteins.

  • 194.
    Fallah, Mahsa
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen: a pleiotropic inflammatory regulator in radiation-induced wound formation and wound repair2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The plasminogen activator (PA) system plays important roles in many physiological and pathological processes, including inflammation and wound healing. Plasmin, the central component of the PA system, is a broad-spectrum serine protease that is derived from its inactive precursor form, plasminogen. The first aim of this thesis was to study the role of plasminogen in the formation of radiation-induced wounds, which are an inflammatory side effect of radiotherapy. The second aim was to investigate the molecular mechanisms behind the potentiating effect of plasminogen in the healing of radiation-induced wounds. The third aim was to explore the therapeutic potential of plasminogen in the healing of radiation-induced wounds.

    Radiation therapy in cancer patients is often limited by side effects such as radiation-induced skin damage (radiodermatitis). The mechanisms behind the formation of radiodermatitis are not fully elucidated, and there are no effective preventive therapies for clinical use. In this study, we show that irradiation of skin in WT (wild-type) mice induces plasminogen accumulation, which is followed by activation of TGF-β (transforming growth factor-beta) signaling and the development of inflammation that leads to skin damage. However, plasminogen-deficient mice and mice lacking PAs were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreases radiodermatitis in WT mice and prevented radiodermatitis in heterozygous mice. Thus, plasmin is required for the formation of radiodermatitis, and inhibition of plasminogen activation might be a novel treatment strategy to reduce or prevent radiodermatitis in patients undergoing radiotherapy.

    Wound healing consists of partially overlapping inflammatory, proliferation, and tissue remodeling phases, and failure to terminate inflammation leads to the formation of chronic wounds. Previous studies by our group have shown that plasminogen is transported to acute wounds by inflammatory cells where it potentiates inflammation and enhances wound healing. Here, we report that plasminogen-deficient mice, which have delayed wound healing, have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialization, indicating inefficient debridement and chronic inflammation. The delayed formation of granulation tissue suggests that fibroblast function is also impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for the resolution of inflammation and the activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing capacity that is comparable to that in WT mice. Therefore, plasminogen might be an important future therapeutic agent for treatment of wounds.

    In radiation-induced wounds, inflammation often cannot resolve and the wounds become chronic and fibrotic. Currently, there is no gold standard for the treatment of radiation-induced wounds. In this study, we have shown that radiation-induced wounds treated with plasminogen healed faster than placebo-treated wounds, had diminished inflammation and granulation tissue formation, and had enhanced re-epithelialization and collagen maturation. Transcriptome analysis showed that plasminogen has a pleiotropic effect on gene expression during wound healing, influencing the expression of 33 genes out of the 84 genes studied. In particular, plasminogen decreased the expression of 11 pro-inflammatory genes early in the healing process. Later, plasminogen decreased WNT (Wingless/Integrated) and TGF-β signaling, as well as the expression of 5 growth factors and 13 factors involved in granulation tissue formation. From the genes downregulated by plasminogen, 19 genes are known to be involved in fibrosis. These results show that in radiation-induced wounds with excessive inflammation and tissue formation plasminogen is able to direct the healing process to a normal outcome without the risk for developing fibrosis. This makes plasminogen an attractive drug candidate for treating radiodermatitis in cancer patients. Taken together, our results indicate that plasminogen is a pleiotropic inflammatory regulator involved in radiation-induced wound formation as well as in wound repair.

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  • 195.
    Fallah, Mahsa
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brodén, Jessica
    Bäckman, Assar
    Lundskog, Bertil
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Johansson, Mikael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Blomqvist, Michael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Liu, Kui
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen activation is required for the development of radiation-induced dermatitis2018In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 9, no 11, article id 1051Article in journal (Refereed)
    Abstract [en]

    Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild type (plg+/+) mice, the plasminogen level increased in the radiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg−/−) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/ mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

     

     

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  • 196.
    Fallah, Mahsa
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viklund, Emil
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bäckman, Assar
    Brodén, Jessica
    Lundskog, Bertil
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Johansson, Mikael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Blomqvist, Michael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Omnio AB, Umeå, Sweden.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen is a master regulator and a potential drug candidate for the healing of radiation wounds2020In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 11, no 3Article in journal (Refereed)
    Abstract [en]

    Around 95% of cancer patients undergoing radiotherapy experience cutaneous side effects, and some develop radiation wounds or fibrosis. Currently, there is no effective treatment for these indications. We show here that plasminogen administration enhanced the healing of radiation wounds via pleiotropic effects on gene expression. Using RNA sequencing, we found that plasminogen downregulated the expression of genes in the TLR, TNF, WNT, MAPK, and TGF-β signaling pathways, and enhanced the anti-inflammatory effect of arachidonic acid, leading to significantly decreased inflammation and improved remodeling of granulation tissue compared with placebo treatment. In addition, plasminogen induced metabolic changes, including decreased glycolysis. Importantly, many of the factors downregulated by plasminogen are pro-fibrotic. Therefore, in radiation wounds with excessive inflammation, plasminogen is able to enhance and redirect the healing process, such that it more closely resembles physiological healing with significantly reduced risk for developing fibrosis. This makes plasminogen an attractive drug candidate for the treatment of radiation wounds in cancer patients.

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  • 197.
    Fallah, Mahsa
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viklund, Emil
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bäckman, Assar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundskog, Bertil
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Johansson, Mikael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Blomqvist, Michael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen enhances the healing of radiation-induced wounds via decreased expression of pro-inflammatory and pro-fibrotic factorsManuscript (preprint) (Other academic)
  • 198. Fallath, Thorya
    et al.
    Kidd, Brendan N.
    Stiller, Jiri
    Davoine, Celine
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Manners, John M.
    Kazan, Kemal
    Schenk, Peer M.
    MEDIATOR18 and MEDIATOR20 confer susceptibility to Fusarium oxysporum in Arabidopsis thaliana2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 4, article id e0176022Article in journal (Refereed)
    Abstract [en]

    The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways.

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  • 199.
    Farag, Salah
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Monika K.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nadeem, Aftab
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Impact of Defective Translocon Assemblies on Hierarchal Yop Effector Translocation by Yersinia pseudotuberculosisManuscript (preprint) (Other academic)
  • 200. Farge, Géraldine
    et al.
    Holmlund, Teresa
    Khvorostova, Julia
    Rofougaran, Reza
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Falkenberg, Maria
    The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities.2008In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 2, p. 393-403Article in journal (Refereed)
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