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  • 101.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Laptenko, Oleg
    Department of Cell Biology, UMDNJ-SOM, Stratford, USA.
    Borukhov, Sergei
    Department of Cell Biology, UMDNJ-SOM, Stratford, USA.
    Chahlafi, Zahra
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Blas-Galindo, Emilio
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Gómez-Puertas, Paulino
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 64, no 3, p. 630-646Article in journal (Refereed)
    Abstract [en]

    The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc-NADH dehydrogenase and the Nar-nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen-sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase sigma(A) holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.

  • 102.
    Cava, Felipe
    et al.
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Zafra, Olga
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    A cytochrome c containing nitrate reductase plays a role in electron transport for denitrification in Thermus thermophilus without involvement of the bc respiratory complex2008In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 2, p. 507-518Article in journal (Refereed)
    Abstract [en]

    The bc(1) respiratory complex III constitutes a key energy-conserving respiratory electron transporter between complex I (type I NADH dehydrogenase) and II (succinate dehydrogenase) and the final nitrogen oxide reductases (Nir, Nor and Nos) in most denitrifying bacteria. However, we show that the expression of complex III from Thermus thermophilus is repressed under denitrification, and that its role as electron transporter is replaced by an unusual nitrate reductase (Nar) that contains a periplasmic cytochrome c (NarC). Several lines of evidence support this conclusion: (i) nitrite and NO are as effective signals as nitrate for the induction of Nar; (ii) narC mutants are defective in anaerobic growth with nitrite, NO and N2O; (iii) such mutants present decreased NADH oxidation coupled to these electron acceptors; and (iv) complementation assays of the mutants reveal that the membrane-distal heme c of NarC was necessary for anaerobic growth with nitrite, whereas the membrane-proximal heme c was not. Finally, we show evidence to support that Nrc, the main NADH oxidative activity in denitrification, interacts with Nar through their respective membrane subunits. Thus, we propose the existence of a Nrc-Nar respiratory super-complex that is required for the development of the whole denitrification pathway in T. thermophilus.

  • 103.
    Cava, Felipe
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Zafra, Olga
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Magalon, Axel
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
    Blasco, Francis
    Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 44, p. 45369-45378Article in journal (Refereed)
    Abstract [en]

    A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.

  • 104.
    Cavka, Adnan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Martin, Carlos
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Alriksson, Bjorn
    Mortsell, Marlene
    Jönsson, Leif J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Techno-economic evaluation of conditioning with sodium sulfite for bioethanol production from softwood2015In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 196, p. 129-135Article in journal (Refereed)
    Abstract [en]

    Conditioning with reducing agents allows alleviation of inhibition of biocatalytic processes by toxic by-products generated during biomass pretreatment, without necessitating the introduction of a separate process step. In this work, conditioning of steam-pretreated spruce with sodium sulfite made it possible to lower the yeast and enzyme dosages in simultaneous saccharification and fermentation (SSF) to 1 g/L and 5 FPU/g WIS, respectively. Techno-economic evaluation indicates that the cost of sodium sulfite can be offset by benefits resulting from a reduction of either the yeast load by 0.68 g/L or the enzyme load by 1 FPU/g WIS. As those thresholds were surpassed, inclusion of conditioning can be justified. Another potential benefit results from shortening the SSF time, which would allow reducing the bioreactor volume and result in capital savings. Sodium sulfite conditioning emerges as an opportunity to lower the financial uncertainty and compensate the overall investment risk for commercializing a softwood-to-ethanol process. (C) 2015 The Authors. Published by Elsevier Ltd.

  • 105.
    Chabes, Anna Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Deoxyribonucleic acid (DNA) carries all the genetic information of a cell. Ribonucleotide reductase (RNR) provides balanced pools of all four dNTPs, the building blocks of DNA. These building blocks are needed during DNA synthesis and repair. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic abnormalities. Because of its central role in dNTP metabolism, RNR is highly regulated on multiple levels.

    The active RNR enzyme consists of two non-identical subunits called proteins R1 and R2. In mammalian cells, during an unperturbed cell cycle, the activity of RNR is highest during S and G2 phases. This is achieved by de novo synthesis of the limiting R2 protein at the onset of S phase, and by controlled degradation of the R2 protein during mitosis.

    This thesis deals with both the S phase-specific transcription of the mouse R2 gene, and the M phase-specific degradation of the mouse R2 protein. Sequence comparison of the mouse R2 promoter to human and guinea pig R2 promoters revealed some conserved elements. These putative regulatory elements were tested in both in vitro and in vivo transcription assays. We demonstrated that the previously identified,

    NF-Y binding CCAAT box is essential for high-level expression from the R2 promoter, but not for its S phase specificity. In addition, the conserved TATA box is dispensable both for basal and S phase-specific R2 transcription as long as the first 17 basepairs of the 5’ untranslated region are present. However, if this 5’ untranslated region is absent, the TATA box is needed for correct initiation of transcription.

    Focusing on the S phase specificity of the R2 gene expression, we demonstrated that the S phase-specific activity of the mouse R2 promoter is dependent on a protein-binding region located ~500 basepairs upstream of the transcription start site and an E2F binding site close to the transcription start site. Deletion of the upstream activating region results in an inactive promoter. In contrast, mutation of the E2F site leads to premature promoter activation in G1 and increased overall promoter activity. However, if the activating mutation of the E2F site is combined with mutation of the upstream activating region, the promoter becomes inactive. These results suggest that the E2F-dependent regulation is important but not sufficient for cell-cycle specific R2 transcription, and that the upstream activating region is crucial for the overall R2 promoter activity.

    In our studies of the M phase-specific R2 degradation, we found that it is dependent on a KEN sequence in the N-terminus of the R2 protein, recognized by the Cdh1-APC complex. Mutating the KEN box stabilizes the R2 protein during mitosis and G1 phase.

    In summary, these studies further extend our understanding of the regulation of the limiting R2 subunit of the enzyme ribonucleotide reductase. The S phase-specific transcription of the R2 gene and the M phase-specific degradation of the R2 protein may serve as important

    mechanisms to protect the cell against unscheduled DNA synthesis.

  • 106.
    Chang, Yanhai
    et al.
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Wang, Xiao
    Department of Galactophore, Shaanxi Provincial Cancer Hospital, Xi’an, PR China.
    Sun, Zhengming
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Jin, Zhankui
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Chen, Ming
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Wang, Xiaoqing
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Lammi, Mikko
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Guo, Xiong
    School of Public Health, Xi’an Jiaotong University Health Science Center, Key Laboratory of Trace Elements and Endemic Diseases, Ministry of Health, Xi’an, China.
    Inflammatory cytokine of IL-1β is involved in T-2 toxin-triggered chondrocyte injury and metabolism imbalance by the activation of Wnt/β-catenin signaling2017In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 91, p. 195-201Article in journal (Refereed)
    Abstract [en]

    Mycotoxin T-2 exerts a causative role in Kashin-Beck disease (KBD) suffering chondrocyte apoptosis and cartilage matrix homeostasis disruption. Recent research corroborated the aberrant levels of pro-inflammatory cytokine IL-1ß in KBD patients and mycotoxin environment. In the present study, we investigated the relevance of IL-1ß in T-2 toxin-evoked chondrocyte cytotoxic injury and aberrant catabolism. High levels of IL-1ß were detected in serum and cartilages from KBD patients and in T-2-stimulated chondrocytes. Moreover, knockdown of IL-1ß antagonized the adverse effects of T-2 on cytotoxic injury by enhancing cell viability and inhibiting apoptosis. However, exogenous supplementation of IL-1β further aggravated cell damage in response to T-2. Additionally, cessation of IL-1β rescued T-2-elicited tilt of matrix homeostasis toward catabolism by elevating the transcription of collagen II and aggrecan, promoting release of sulphated glycosaminoglycans (sGAG) and TIMP1, and suppressing matrix metalloproteinases production including MMP-1, MMP-3 and MMP-13. Conversely, IL-1β stimulation deteriorated T-2-induced disruption of matrix metabolism balance toward catabolism. Mechanistic analysis found the high activation of Wnt/β-catenin in KBD patients and chondrocytes upon T-2. Furthermore, this activation was mitigated after IL-1β inhibition, but further enhanced following IL-1β precondition. Importantly, blocking this pathway by transfection with β-catenin alleviated the adverse roles of IL-1β on cytotoxic injury and metabolism disorders under T-2 conditioning. Together, this study elucidates a new insight into how T-2 deteriorates the pathological progression of KBD by regulating inflammation-related pathways, indicating a promising anti-inflammation strategy for KBD therapy.

  • 107.
    Charpentier, Emmanuelle
    et al.
    Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology.
    Hess, Wolfgang R
    RNA in bacteria: biogenesis, regulatory mechanisms and functions2015In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, no 3, p. 277-279Article in journal (Refereed)
  • 108.
    Chautard, Hélène
    et al.
    Biométhodes SA, Evry, France.
    Blas-Galindo, Emilio
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Menguy, Thierry
    Biométhodes SA, Evry, France.
    Grand'Moursel, Laure
    Biométhodes SA, Evry, France.
    Cava, Felipe
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Berenguer, José
    CBM ‘Severo Ochoa’ CSIC-UAM, Madrid, Spain.
    Delcourt, Marc
    Biométhodes SA, Evry, France.
    An activity-independent selection system of thermostable protein variants2007In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 4, no 11, p. 919-921Article in journal (Refereed)
    Abstract [en]

    We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

  • 109. Checa, A.
    et al.
    Idborg, H.
    Zandian, A.
    Sar, D. Garcia
    Surowiec, Izabella
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Svenungsson, E.
    Jakobsson, P-J
    Nilsson, P.
    Gunnarsson, I.
    Wheelock, C. E.
    Dysregulations in circulating sphingolipids associate with disease activity indices in female patients with systemic lupus erythematosus: a cross-sectional study2017In: Lupus, ISSN 0961-2033, E-ISSN 1477-0962, Vol. 26, no 10, p. 1023-1033Article in journal (Refereed)
    Abstract [en]

    Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus.

    Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease.

    Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C-16:0-ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C-16:0-ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C-16:0- and C-24:1-hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment.

    Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated with disease activity. This study demonstrates the utility of simultaneously targeting multiple components of a pathway to establish disease associations.

  • 110.
    Chen, Changchun
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Anderson, James T
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Unexpected accumulation of ncm5U and ncm5s2U in a trm9 mutant suggests an additional step in the synthesis of mcm5U and mcm5s2U.2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 6, p. e20783-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm(5)U), 5-carbamoylmethyl-2'-O-methyluridine (ncm(5)Um), 5-methoxycarbonylmethyl-uridine (mcm(5)U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The formation of mcm(5) and ncm(5) side chains involves a complex pathway, where the last step in formation of mcm(5) is a methyl esterification of cm(5) dependent on the Trm9 and Trm112 proteins.

    METHODOLOGY AND PRINCIPAL FINDINGS: Both Trm9 and Trm112 are required for the last step in formation of mcm(5) side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm(5)U or mcm(5)s(2)U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm(5)U and mcm(5)s(2)U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm(5)U and ncm(5)s(2)U, not the expected cm(5)U and cm(5)s(2)U.

    CONCLUSIONS: Trm9p and Trm112p function together at the final step in formation of mcm(5)U in tRNA by using the intermediate cm(5)U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm(5)U and ncm(5)s(2)U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm(5) side chain. We propose two alternative explanations for this observation. One is that the intermediate cm(5)U is generated from ncm(5)U by a yet unknown mechanism and the other is that cm(5)U is formed before ncm(5)U and mcm(5)U.

  • 111.
    Chen, Changchun
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Eliasson, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Rydén, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elongator Complex Influences Telomeric Gene Silencing and DNA Damage Response by Its Role in Wobble Uridine tRNA Modification2011In: PLoS genetics, ISSN 1553-7404, Vol. 7, no 9, p. e1002258-Article in journal (Refereed)
    Abstract [en]

    Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm(5)U(34)), 5-methoxycarbonylmethyluridine (mcm(5)U(34)), and 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys) (s(2) ) (UUU), tRNA(Gln) (s(2) ) (UUG), and tRNA(Glu) (s(2) ) (UUC), which in a wild-type background contain the mcm(5)s(2)U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U(34). Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm(5)s(2)U nucleoside in tRNA(Lys) (mcm(5) (s(2) ) (UUU) ), tRNA(Gln) (mcm(5) (s(2) ) (UUG) ), and tRNA(Glu) (mcm(5) (s(2) ) (UUC) ). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U(34) are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.

  • 112.
    Chen, Eefei
    et al.
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Christiansen, Alexander
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wang, Qian
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Cheung, Margaret S
    Department of Physics, University of Houston, Houston, Texas 77204, United States.
    Kliger, David S
    Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 49, p. 9836-9845Article in journal (Refereed)
    Abstract [en]

    Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

  • 113.
    Chen, Peng
    Umeå University, Faculty of Medicine, Molecular Biology.
    Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described.

    A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C.

    cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent.

    During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

  • 114. Chen, Peng
    et al.
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Zheng, Bo
    Transfer RNA modifications and genes for modifying enzymes in Arabidopsis thaliana2010In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 10, article id 201Article in journal (Refereed)
    Abstract [en]

    Background: In all domains of life, transfer RNA (tRNA) molecules contain modified nucleosides. Modifications to tRNAs affect their coding capacity and influence codon-anticodon interactions. Nucleoside modification deficiencies have a diverse range of effects, from decreased virulence in bacteria, neural system disease in human, and gene expression and stress response changes in plants. The purpose of this study was to identify genes involved in tRNA modification in the model plant Arabidopsis thaliana, to understand the function of nucleoside modifications in plant growth and development. Results: In this study, we established a method for analyzing modified nucleosides in tRNAs from the model plant species, Arabidopsis thaliana and hybrid aspen (Populus tremula x tremuloides). 21 modified nucleosides in tRNAs were identified in both species. To identify the genes responsible for the plant tRNA modifications, we performed global analysis of the Arabidopsis genome for candidate genes. Based on the conserved domains of homologs in Sacccharomyces cerevisiae and Escherichia coli, more than 90 genes were predicted to encode tRNA modifying enzymes in the Arabidopsis genome. Transcript accumulation patterns for the genes in Arabidopsis and the phylogenetic distribution of the genes among different plant species were investigated. Transcripts for the majority of the Arabidopsis candidate genes were found to be most abundant in rosette leaves and shoot apices. Whereas most of the tRNA modifying gene families identified in the Arabidopsis genome was found to be present in other plant species, there was a big variation in the number of genes present for each family. Through a loss of function mutagenesis study, we identified five tRNA modification genes (AtTRM10, AtTRM11, AtTRM82, AtKTI12 and AtELP1) responsible for four specific modified nucleosides (m1G, m2G, m7G and ncm5U), respectively (two genes: AtKTI12 and AtELP1 identified for ncm5U modification). The AtTRM11 mutant exhibited an early-flowering phenotype, and the AtELP1 mutant had narrow leaves, reduced root growth, an aberrant silique shape and defects in the generation of secondary shoots. Conclusions: Using a reverse genetics approach, we successfully isolated and identified five tRNA modification genes in Arabidopsis thaliana. We conclude that the method established in this study will facilitate the identification of tRNA modification genes in a wide variety of plant species.

  • 115.
    Chen, Xi
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Wu, Yao-Wen
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Tunable and Photoswitchable Chemically Induced Dimerization for Chemo-optogenetic Control of Protein and Organelle Positioning2018In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 57, no 23, p. 6796-6799Article in journal (Refereed)
    Abstract [en]

    The spatiotemporal dynamics of proteins and organelles play an important role in controlling diverse cellular processes. Optogenetic tools using photosensitive proteins and chemically induced dimerization (CID), which allow control of protein dimerization, have been used to elucidate the dynamics of biological systems and to dissect the complicated biological regulatory networks. However, the inherent limitations of current optogenetic and CID systems remain a significant challenge for the fine-tuning of cellular activity at precise times and locations. Herein, we present a novel chemo-optogenetic approach, photoswitchable chemically induced dimerization (psCID), for controlling cellular function by using blue light in a rapid and reversible manner. Moreover, psCID is tunable; that is, the dimerization and dedimerization degrees can be fine-tuned by applying different doses of illumination. Using this approach, we control the localization of proteins and positioning of organelles in live cells with high spatial (μm) and temporal (ms) precision.

  • 116. Cheng, Fang
    et al.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Centre for Heart Lung Innovation, St. Paul ’ s Hospital, Vancouver, BC, Canada V6Z 1Y6; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6Z 1Y6.
    Mohanasundaram, Ponnuswamy
    Lindstrom, Michelle
    Ivaska, Johanna
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Eriksson, John E.
    Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-beta-Slug signaling2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 30, p. E4320-E4327Article in journal (Refereed)
    Abstract [en]

    Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-beta 1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM-/-) wounds. Correspondingly, VIM-/- wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM-/- wounds. Vimentin reconstitution in VIM-/- fibroblasts restored both their proliferation and TGF-beta 1 production. Similarly, restoring paracrine TGF-beta-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-beta 1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation.

  • 117.
    Cheregi, Otilia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wagner, Raik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Insights into the Cyanobacterial Deg/HtrA Proteases2016In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 7, article id 694Article in journal (Refereed)
    Abstract [en]

    Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serinetype ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior.

  • 118. Chereji, Razvan V.
    et al.
    Bharatula, Vasudha
    Elfving, Nils
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Miriam
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozov, Alexandre V.
    Broach, James R.
    Björklund, Stefan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 15, p. 8806-8821Article in journal (Refereed)
    Abstract [en]

    Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.

  • 119.
    Chilkova, Olga
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Functional and structural properties of eukaryotic DNA polymerase epsilon2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In eukaryotes there are three DNA polymerases which are essential for the replication of chromosomal DNA: DNA polymerase alpha (Pol alpha), DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon). In vitro studies of viral DNA replication showed that Pol alpha and Pol delta are sufficient for DNA replication on both leading and lagging DNA strands, thus leaving the function of Pol epsilon unknown. The low abundance and the reported protease sensitivity of Pol epsilon were holding back biochemical studies of the enzyme. The aim of this study was to characterize the structural and functional properties of eukaryotic Pol epsilon.

    We first developed a protocol for over-expression and purification of Pol epsilon from the yeast Saccharomyces cerevisiae. Pol epsilon consists of four subunits: Pol2 (catalytic subunit), Dpb2, Dpb3 and Dpb4. This four-subunit complex was purified to homogeneity by conventional chromatography and the subunit stoichiometry of purified Pol epsilon was estimated from colloidal coomassie-stained gels to be 1:1:1:1. The quaternary structure was determined by sedimentation velocity and gel filtration experiments. Molecular mass (371 kDa) was calculated from the experimentally determined Stokes radius (74.5 Å) and sedimentation coefficient (11.9 S) and was in good agreement with a theoretical molecular mass calculated for a heterotetramer (379 kDa). Analytical sedimentation equilibrium ultracentrifugation experiments supported the proposed heterotetrameric structure of Pol epsilon.

    By cryo-electron microscopy and single-particle image analysis we determined the structure of Saccharomyces cerevisiae Pol epsilon to 20-Å resolution. The four-subunit complex was found to consist of a globular domain, comprising the Pol2 subunit, flexibly connected to an elongated domain, including Dpb2, Dpb3 and Dpb4 subunits. We found that Pol epsilon requires a minimal length of 40 base pairs of primer-template duplex to be processive. This length corresponds to the dimensions of the elongated domain.

    To characterize the fidelity by which Pol epsilon synthesizes DNA, we purified wild type and exonuclease-deficient Pol epsilon. Wild type Pol epsilon synthesizes DNA with a very high accuracy. Analysis of the exonuclease-deficient Pol epsilon showed that Pol epsilon proofreads more than 90% of the errors made by its polymerase activity. Exonuclease-deficient Pol epsilon was shown to have a specific spectrum of errors not seen in other DNA polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP and C-dTTP mispairs. This unique error specificity and amino acid sequence alignment suggest that the structure of the polymerase active site of Pol epsilon differs from those of other members of B family DNA polymerases.

    With recombinant proteins and circular single-stranded DNA templates, we partially reconstituted DNA replication in vitro, in which we challenged Pol epsilon and Pol delta in side-by-side comparisons regarding functional assays for polymerase activity and processivity, as well as physical interactions with nucleic acids and PCNA. We found that Pol epsilon activity and “on-DNA” PCNA interactions are dependent on RPA-coated template DNA. By the surface plasmon resonance technique, we showed that Pol epsilon has a high affinity for DNA and low affinity for immobilized PCNA. By contrast, Pol delta was found to have low affinity for DNA and high affinity for PCNA. We suggest that a possible function of RPA is to regulate down the DNA synthesis through Pol epsilon, and that the mechanism by which Pol epsilon and Pol delta load onto the template is different due to different properties of the interaction with DNA and PCNA.

  • 120.
    Chorell, Elin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Svensson, Michael
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Moritz, Thomas
    Swedish University of Agricultural Sciences.
    Antti, Henrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Physical fitness level is reflected by alterations in the human plasma metabolome2012In: Molecular BioSystems, ISSN 1742-206X, Vol. 8, no 4, p. 1187-1196Article in journal (Refereed)
    Abstract [en]

    An excessive energy intake combined with a low level of physical activity induces detrimental processes involved in disease development, e.g. type 2 diabetes and cardiovascular disease. However the underlying mechanisms for regulation of metabolic capacity and fitness status remain unclear. Metabolomics involves global studies of the metabolic reactions in an organism or cell. Thus hypotheses regarding biochemical events can be generated to increase the understanding of disease development and thereby aid in the development of novel treatments or preventions. We present the first standardized intervention study focusing on characterizing the human metabolome in relation to moderate differences in cardiorespiratory fitness. Gas chromatography-time of flight/mass spectrometry (GC-TOF/MS) was used to characterize 460 plasma samples from 27 individuals divided into two groups based on physical fitness level (VO2max). Multi- and univariate between group comparisons based on 197 metabolites were carried out in samples collected at rest prior to any intervention, over time following a nutritional load or a standardized exercise scheme, with and without nutritional load. We detected decreased levels of gamma-tocopherol (GT), a vitamin E isomer, in response to a high fitness level, whereas the opposite was seen for the alpha isomer (AT). In addition, the high fitness level was associated with elevated ω3-PUFA (DHA, 22:6ω3) and a decrease in ω6-PUFA (18:2ω6) as well as in saturated (16:0, 18:0), monounsaturated (18:1) and trans (16:1) fatty acids. We thus hypothesize that high fitness status induces an increased cardiorespiratory inflammatory and antioxidant defense system, more prone to deal with the inflammatory response following exercise and nutrition intake.

  • 121. Coenye, Tom
    et al.
    Van Dijck, Patrick
    Bjarnsholt, Thomas
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Microbial biofilms - the coming of age of a research field2014In: Pathogens and Disease, ISSN 2049-632X, Vol. 70, no 3, p. 203-204Article in journal (Other academic)
  • 122.
    Comstedt, Pär
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Biology of Borrelia garinii Spirochetes2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Lyme borreliosis is a tick-transmitted infectious disease. The causative agents are spiral-shaped bacteria and the most common sign of infection is a skin rash at the site of the tick bite. If not treated with antibiotics, the bacteria can disseminate and cause a variety of different manifestations including arthritis, carditis or neurological problems. The disease is a zoonosis and the bacteria are maintained in nature by different vertebrate reservoir host animals. In Europe, three different Borrelia genospecies cause Lyme borreliosis: B. burgdorferi, B. afzelii and B. garinii. The latter depends in part on birds as its reservoir host. B. garinii bacteria have been found in a marine enzootic infection cycle worldwide and also among terrestrial birds. This thesis suggests that passerine birds and seabirds constitute an important reservoir for B. garinii bacteria also with clinical importance. We have found bacteria very similar to Lyme borreliosis causing isolates in ticks infesting migrating passerine birds. The birds not only transport infected ticks, but are competent reservoir hosts, as measured by their ability to infect naïve ticks. Their role as a reservoir host is dependent on their foraging behavior, where ground-dwelling birds are of greater importance than other species. When comparing B. garinii isolates from Europe, the Arctic and North Pacific, and including isolates from seabirds, passerine birds, Ixodes ricinus ticks and Lyme borreliosis patients, we found that phylogenetic grouping was not necessarily dependent on geographical or biological origin. B. garinii from seabirds were very heterogeneous and found in all different groups. Therefore, the marine and the terrestrial infection cycles are likely to overlap. This was supported by the fact that B. garinii isolated from seabirds can establish a long-term infection in mice. Bacteria from the genospecies B. garinii are overrepresented among neuroborreliosis patients. Interestingly, many clinical B. garinii isolates are sensitive to human serum and have shown weak binding to the complement inhibitor protein factor H. By transforming a serum-sensitive B. garinii isolate with a shuttle vector containing the gene for the factor H binding protein OspE from complement-resistant B. burgdorferi, serum resistance could be increased. In addition, neurovirulent B. garinii strains recently isolated from neuroborreliosis patients were shown to express a factor H binding protein, not found in bacteria that had been kept in culture for a long time. This protein may contribute to the virulence of neuroborreliosis-causing B. garinii strains. When testing B. garinii isolates from Lyme borreliosis patients and seabirds for resistance to human serum, all members of the latter group were sensitive to even low levels of serum. This suggests that seabird isolates are not capable of infecting humans. In agreement with this, B. garinii isolated from seabirds do not appear to bind human factor H.

  • 123.
    Conaway, H. Herschel
    et al.
    Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas.
    Pirhayati, Amir
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Persson, Emma
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Pettersson, Ulrika
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Pharmacology.
    Svensson, Olle
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Lindholm, Catharina
    Center for Bone and Arthritis Research at the Institute for Medicine, Sahlgrenska Academy at the University of Gothenburg.
    Henning, Petra
    Center for Bone and Arthritis Research at the Institute for Medicine, Sahlgrenska Academy at the University of Gothenburg.
    Tuckermann, Jan
    Tissue-specific Hormone Action, Leibniz Institute for Age Research, Fritz Lipmann Institute, D-07745 Jena, Germany.
    Lerner, Ulf H.
    Umeå University, Faculty of Medicine, Department of Odontology, Molecular Periodontology.
    Retinoids Stimulate Periosteal Bone Resorption by Enhancing the Protein RANKL: a Response Inhibited by Monomeric Glucocorticoid Receptor2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, p. 31425-31436Article in journal (Refereed)
    Abstract [en]

    Increased vitamin A (retinol) intake has been suggested to increase bone fragility. In the present study, we investigated effects of retinoids on bone resorption in cultured neonatal mouse calvarial bones and their interaction with glucocorticoids (GC). All-trans-retinoic acid (ATRA), retinol, retinalaldehyde, and 9-cis-retinoic acid stimulated release of (45)Ca from calvarial bones. The resorptive effect of ATRA was characterized by mRNA expression of genes associated with osteoclast differentiation, enhanced osteoclast number, and bone matrix degradation. In addition, the RANKL/OPG ratio was increased by ATRA, release of (45)Ca stimulated by ATRA was blocked by exogenous OPG, and mRNA expression of genes associated with bone formation was decreased by ATRA. All retinoid acid receptors (RAR alpha/beta/gamma) were expressed in calvarial bones. Agonists with affinity to all receptor subtypes or specifically to RAR alpha enhanced the release of (45)Ca and mRNA expression of Rankl, whereas agonists with affinity to RAR beta/gamma or RAR gamma had no effects. Stimulation of Rankl mRNA by ATRA was competitively inhibited by the RAR alpha antagonist GR110. Exposure of calvarial bones to GC inhibited the stimulatory effects of ATRA on 45Ca release and Rankl mRNA and protein expression. This inhibitory effect was reversed by the glucocorticoid receptor (GR) antagonist RU 486. Increased Rankl mRNA stimulated by ATRA was also blocked by GC in calvarial bones from mice with a GR mutation that blocks dimerization (GR(dim) mice). The data suggest that ATRA enhances periosteal bone resorption by increasing the RANKL/OPG ratio via RAR alpha receptors, a response that can be inhibited by monomeric GR.

  • 124. Conn, Vanessa M.
    et al.
    Hugouvieux, Veronique
    Nayak, Aditya
    Conos, Stephanie A.
    Capovilla, Giovanna
    Cildir, Gokhan
    Jourdain, Agnes
    Tergaonkar, Vinay
    Schmid, Markus
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Zubieta, Chloe
    Conn, Simon J.
    A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation2017In: Nature Plants, ISSN 2055-026X, Vol. 3, no 5, article id 17053Article in journal (Refereed)
    Abstract [en]

    Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life(1), yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA: DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS(2-4). This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.

  • 125. Cornelis, M C
    et al.
    Byrne, E M
    Esko, T
    Nalls, M A
    Ganna, A
    Paynter, N
    Monda, K L
    Amin, N
    Fischer, K
    Renstrom, F
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Nutritional Research. Umeå University, Faculty of Medicine, Department of Biobank Research.
    Ngwa, J S
    Huikari, V
    Cavadino, A
    Nolte, I M
    Teumer, A
    Yu, K
    Marques-Vidal, P
    Rawal, R
    Manichaikul, A
    Wojczynski, M K
    Vink, J M
    Zhao, J H
    Burlutsky, G
    Lahti, J
    Mikkilä, V
    Lemaitre, R N
    Eriksson, J
    Musani, S K
    Tanaka, T
    Geller, F
    Luan, J
    Hui, J
    Mägi, R
    Dimitriou, M
    Garcia, M E
    Ho, W-K
    Wright, M J
    Rose, L M
    Magnusson, P K E
    Pedersen, N L
    Couper, D
    Oostra, B A
    Hofman, A
    Ikram, M A
    Tiemeier, H W
    Uitterlinden, A G
    van Rooij, F J A
    Barroso, I
    Johansson, Ingegerd
    Umeå University, Faculty of Medicine, Department of Odontology. Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Nutritional Research. Umeå University, Faculty of Medicine, Department of Biobank Research.
    Xue, L
    Kaakinen, M
    Milani, L
    Power, C
    Snieder, H
    Stolk, R P
    Baumeister, S E
    Biffar, R
    Gu, F
    Bastardot, F
    Kutalik, Z
    Jacobs, D R
    Forouhi, N G
    Mihailov, E
    Lind, L
    Lindgren, C
    Michaëlsson, K
    Morris, A
    Jensen, M
    Khaw, K-T
    Luben, R N
    Wang, J J
    Männistö, S
    Perälä, M-M
    Kähönen, M
    Lehtimäki, T
    Viikari, J
    Mozaffarian, D
    Mukamal, K
    Psaty, B M
    Döring, A
    Heath, A C
    Montgomery, G W
    Dahmen, N
    Carithers, T
    Tucker, K L
    Ferrucci, L
    Boyd, H A
    Melbye, M
    Treur, J L
    Mellström, D
    Hottenga, J J
    Prokopenko, I
    Tönjes, A
    Deloukas, P
    Kanoni, S
    Lorentzon, M
    Houston, D K
    Liu, Y
    Danesh, J
    Rasheed, A
    Mason, M A
    Zonderman, A B
    Franke, L
    Kristal, B S
    Karjalainen, J
    Reed, D R
    Westra, H-J
    Evans, M K
    Saleheen, D
    Harris, T B
    Dedoussis, G
    Curhan, G
    Stumvoll, M
    Beilby, J
    Pasquale, L R
    Feenstra, B
    Bandinelli, S
    Ordovas, J M
    Chan, A T
    Peters, U
    Ohlsson, C
    Gieger, C
    Martin, N G
    Waldenberger, M
    Siscovick, D S
    Raitakari, O
    Eriksson, J G
    Mitchell, P
    Hunter, D J
    Kraft, P
    Rimm, E B
    Boomsma, D I
    Borecki, I B
    Loos, R J F
    Wareham, N J
    Vollenweider, P
    Caporaso, N
    Grabe, H J
    Neuhouser, M L
    Wolffenbuttel, B H R
    Hu, F B
    Hyppönen, E
    Järvelin, M-R
    Cupples, L A
    Franks, Paul W
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine. Department of Nutrition, Harvard School of Public Health, Boston, MA, USA; Lund Univ, Dept Clin Sci, Malmo, Sweden.
    Ridker, P M
    van Duijn, C M
    Heiss, G
    Metspalu, A
    North, K E
    Ingelsson, E
    Nettleton, J A
    van Dam, R M
    Chasman, D I
    Genome-wide meta-analysis identifies six novel loci associated with habitual coffee consumption2015In: Molecular Psychiatry, ISSN 1359-4184, E-ISSN 1476-5578, Vol. 20, no 5, p. 647-656Article in journal (Refereed)
    Abstract [en]

    Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91 462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10(-8)).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee.

  • 126. Cornes, Michael P.
    et al.
    Church, Stephen
    van Dongen-Lases, Edmee
    Grankvist, Kjell
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Guimaraes, Joao T.
    Ibarz, Mercedes
    Kovalevskaya, Svetlana
    Kristensen, Gunn B. B.
    Lippi, Giuseppe
    Nybo, Mads
    Sprongl, Ludek
    Sumarac, Zorica
    Simundic, Ana-Maria
    The role of European Federation of Clinical Chemistry and Laboratory Medicine Working Group for Preanalytical Phase in standardization and harmonization of the preanalytical phase in Europe2016In: Annals of Clinical Biochemistry, ISSN 0004-5632, E-ISSN 1758-1001, Vol. 53, no 5, p. 539-547Article, review/survey (Refereed)
    Abstract [en]

    Patient safety is a leading challenge in healthcare and from the laboratory perspective it is now well established that preanalytical errors are the major contributor to the overall rate of diagnostic and therapeutic errors. To address this, the European Federation of Clinical Chemistry and Laboratory Medicine Working Group for Preanalytical Phase (EFLM WG-PRE) was established to lead in standardization and harmonization of preanalytical policies and practices at a European level. One of the key activities of the WG-PRE is the organization of the biennial EFLM-BD conference on the preanalytical phase to provide a forum for National Societies (NS) to discuss their issues. Since 2012, a year after the first Preanalytical phase conference, there has been a rapid growth in the number of NS with a working group engaged in preanalytical phase activities and there are now at least 19 countries that have one. As a result of discussions with NS at the third conference held in March 2015 five key areas were identified as requiring harmonisation. These were test ordering, sample transport and storage, patient preparation, sampling procedures and management of unsuitable specimens. The article below summarises the work that has and will be done in these areas. The goal of this initiative is to ensure the EFLM WG-PRE produces work that meets the needs of the European laboratory medicine community. Progress made in the identified areas will be updated at the next preanalytical phase conference and show that we have produced guidance that has enhanced standardisation in the preanalytical phase and improved patient safety throughout Europe.

  • 127.
    Costa, Tiago
    et al.
    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).
    Amer, Ayad
    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).
    Farag, Salah
    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).
    Wolf-Watz, Hans
    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). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fällman, Maria
    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).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    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).
    Francis, Matthew
    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).
    Type III secretion translocon assemblies that attenuate Yersinia virulence2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 7, p. 1088-1110Article in journal (Refereed)
    Abstract [en]

    Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD(A263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

  • 128.
    Courtois-Moreau, Charleen, Laetitia
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre.
    Programmed Cell Death in Xylem Development2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Concerns about climate changes and scarcity of fossil fuels are rising. Hence wood is becoming an attractive source of renewable energy and raw material and these new dimensions have prompted increasing interest in wood formation in trees, in both the scientific community and wider public. In this thesis, the focus is on a key process in wood development: programmed cell death (PCD) in the development of xylem elements. Since secondary cell wall formation is dependent, inter alia, upon the life time of xylem elements, the qualitative features of wood will be affected by PCD in xylem, about which there is little information.

    This thesis focuses on the anatomical, morphological and transcriptional features of PCD during xylem development in both the stem of hybrid aspen, Populus tremula (L.) x tremuloides (Michx.) and the hypocotyl of the herbaceous model system Arabidopsis thaliana (L. Heynh.). In Populus, the progressive removal of organelles from the cytoplasm before the time of death (vacuolar bursts) and the slowness of the cell death process, illustrated by DNA fragmentation assays (such as TUNEL and Comet assays), have been ascertained in the xylem fibres by microscopic analyses. Furthermore, candidate genes for the regulation of fibre cell death were identified either from a Populus EST library obtained from woody tissues undergoing fibre cell death or from microarray experiments in Populus stem, and further assessed in an in silico comparative transcriptomic analysis of Arabidopsis thaliana. These candidate genes were either putative novel regulators of fibre cell death or members of previously described families of cell death-related genes, such as autophagy-related genes. The induction of the latter and the previous microscopic observations suggest the importance of autophagy in the degradation of the cytoplasmic contents specifically in the xylem fibres. Vacuolar bursts in the vessels were the only previously described triggers of PCD in the xylem, which induce the very rapid degradation of the nuclei and surrounding cytoplasmic contents, therefore unravelling a unique previously unrecorded type of PCD in the xylem fibres, principally involving autophagy. Arabidopsis is an attractive alternative model plant for exploring some aspects of wood formation, such as the characterisation of negative regulators of PCD. Therefore, the anatomy of Arabidopsis hypocotyls was also investigated and the ACAULIS5 (ACL5) gene, encoding an enzyme involved in polyamine biosynthesis, was identified as a key regulator of xylem specification, specifically in the vessel elements, though its negative effect on the cell death process.

    Taken together, PCD in xylem development seems to be a highly specific process, involving unique cell death morphology and molecular machinery. In addition, the technical challenges posed by the complexity of the woody tissues examined highlighted the need for specific methods for assessing PCD and related phenomena in wood.

  • 129.
    Crawford, Tim
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Lehotai, Nóra
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Strand, Åsa
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The role of retrograde signals during plant stress responses2018In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 69, no 11, p. 2783-2795Article, review/survey (Refereed)
    Abstract [en]

    Chloroplast and mitochondria not only provide the energy to the plant cell but due to the sensitivity of organellar processes to perturbations caused by abiotic stress, they are also key cellular sensors of environmental fluctuations. Abiotic stresses result in reduced photosynthetic efficiency and thereby reduced energy supply for cellular processes. Thus, in order to acclimate to stress, plants must re-program gene expression and cellular metabolism to divert energy from growth and developmental processes to stress responses. To restore cellular energy homeostasis following exposure to stress, the activities of the organelles must be tightly co-ordinated with the transcriptional re-programming in the nucleus. Thus, communication between the organelles and the nucleus, so-called retrograde signalling, is essential to direct the energy use correctly during stress exposure. Stress-triggered retrograde signals are mediated by reactive oxygen species and metabolites including beta-cyclocitral, MEcPP (2-C-methyl-D-erythritol 2,4-cyclodiphosphate), PAP (3'-phosphoadenosine 5'-phosphate), and intermediates of the tetrapyrrole biosynthesis pathway. However, for the plant cell to respond optimally to environmental stress, these stress-triggered retrograde signalling pathways must be integrated with the cytosolic stress signalling network. We hypothesize that the Mediator transcriptional co-activator complex may play a key role as a regulatory hub in the nucleus, integrating the complex stress signalling networks originating in different cellular compartments.

  • 130. Crona, Mikael
    et al.
    Torrents, Eduard
    Rohr, Asmund K.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Furrer, Ernst
    Tomter, Ane B.
    Andersson, K. Kristoffer
    Sahlin, Margareta
    Sjoberg, Britt-Marie
    NrdH-Redoxin Protein Mediates High Enzyme Activity in Manganese-reconstituted Ribonucleotide Reductase from Bacillus anthracis2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 38, p. 33053-33060Article in journal (Refereed)
    Abstract [en]

    Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

  • 131. Crowe-McAuliffe, Caillan
    et al.
    Graf, Michael
    Huter, Paul
    Takada, Hiraku
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Abdelshahid, Maha
    Novácek, Jirí
    Murina, Victoriia
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Atkinson, Gemma C.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
    Wilson, Daniel N.
    Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 36, p. 8978-8983Article in journal (Refereed)
    Abstract [en]

    Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ(2) mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).

  • 132.
    Croxatto, Antony
    Umeå University, Faculty of Medicine, Molecular Biology.
    VanT, a central regulator of quorum sensing signalling in Vibrio anguillarum2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Many bacteria produce signal molecules that serve in a cell-to-cell communication system termed quorum sensing. This signalling system allows a bacterial population to co-ordinately regulate functions according to their cell number in a defined environment. As bacterial growth progresses towards the stationary phase, signalling molecules accumulate in the growth medium and, above a certain threshold level, regulate the expression of genes involved in diverse functions. Most of the functions monitored by quorum sensing are most beneficial when they are performed as a population than by single cells, such as virulence factor production, biofilm formation, conjugation and bioluminescence.

    Vibrio anguillarum is a bacterial pathogen that causes terminal hemorrhagic septicaemia in marine fish. V. anguillarum possesses multiple quorum sensing circuits similar to the LuxI/LuxR and the V. harveyi-type systems. In this study, a characterisation of the quorum sensing-regulated transcriptional activator VanT was made. VanT belongs to the V. harveyi LuxR family of transcriptional regulators, which play a central role in quorum sensing signalling in Vibrio species. VanT was shown to regulate serine, metalloprotease, pigment, exopolysaccharide (EPS) and biofilm production. VanT repressed an EPS locus that plays a critical role in bacterial colonization of the fish integument and virulence.

    The V. harveyi-like quorum sensing systems were shown to limit rather than induce vanT expression throughout growth in V. anguillarum. In contrast to homologous proteins in other Vibrio spp., the quorum sensing phosphorelay protein VanU and the response regulator VanO had antagonistic roles in the regulation of vanT expression. Unlike other members of the luxR family, vanT was expressed at low cell density and no significant induction due to quorum sensing regulation was seen.

    Interestingly, VanT expression was induced by the alternative sigma factor RpoS as the cells entered stationary phase. RpoS was shown to regulate VanT expression post-transcriptionally by promoting vanT mRNA stability. VanT and RpoS were important for bacterial survival under stress conditions, indicating that VanT is likely an essential factor of V. anguillarum stress response.

  • 133. Cruz-Ramírez, Alfredo
    et al.
    Díaz-Triviño, Sara
    Blilou, Ikram
    Grieneisen, Verônica A.
    Sozzani, Rosangela
    Zamioudis, Christos
    Miskolczi, Pál
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Forest Genetics and Plant Physiology, Umeå Plant Science Center, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Nieuwland, Jeroen
    Benjamins, René
    Dhonukshe, Pankaj
    Caballero-Pérez, Juan
    Horvath, Beatrix
    Long, Yuchen
    Mähönen, Ari Pekka
    Zhang, Hongtao
    Xu, Jian
    Murray, James A. H.
    Benfey, Philip N.
    Bako, Laszlo
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Forest Genetics and Plant Physiology, Umeå Plant Science Center, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Marée, Athanasius F. M.
    Scheres, Ben
    A Bistable Circuit Involving SCARECROW-RETINOBLASTOMA Integrates Cues to Inform Asymmetric Stem Cell Division2012In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 150, no 5, p. 1002-1015Article in journal (Refereed)
    Abstract [en]

    In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOM-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.

  • 134.
    Dahrendorf, Julia
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Analysis of nitrogen utilization capability during proliferation and maturation of Norway spruce (Picea abies L. Karst) somatic embryogenesis2014Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Around 70 % of the standing trees in northern Europe are conifers, with Norway spruce being most important. To meet future wood demands, vegetative propagation methods are offering a flexible and effective way to multiply superior genotypes. The development of nitrogen metabolism during embryogenesis is not well understood and only few studies cover conifers. Norway spruce plants prefer ammonium over nitrate as an inorganic nitrogen source. However, the proliferation of somatic embryo cultures requires organic nitrogen, and ammonium nitrate as sole nitrogen source limits somatic embryo development. This raises the question how nitrogen utilization capability advances throughout the embryo development and plant formation in Norway spruce and suggests a developmental switch in nitrogen utilization capability before the plant is fully developed. Of special interest in this context is the development and activity of three key enzymes of nitrogen metabolism: nitrate reductase (NR), glutamine synthetase (GS) and arginase.

    The aim of this study was to investigate the importance of L-glutamine as an organic nitrogen source and its impact on these key enzymes of nitrogen metabolism in the proliferation and maturation stage of Norway spruce somatic embryogenesis. Therefore media with modified nitrogen sources have been used to study the effects of presence and withdrawal of L-glutamine. Pro-embryogenic masses (PEMs) grown with L-glutamine (Gln) or L-glutamine and nitrate (Gln + NO3) showed a strongly improved proliferation rate in comparison to PEMs grown on ammonium nitrate (NH4NO3). Interestingly, GS and NR were inactive enzymatically in PEMs. Arginase activity was observed, and was unaffected by the presence or absence of L-glutamine. For analyzing the importance of L-glutamine as an organic nitrogen source during maturation, somatic embryos have been generated on media with modified nitrogen sources that included also autoclaved casein hydrolysate, an amino-acid mixture that lacks L-glutamine after autoclaving. Somatic embryos matured furthest regarding size and cotyledon development on Gln + NO3. Maturation on NH4NO3 resulted in well-developed cotyledonary stage somatic embryos that were smaller in size than in the presence of L-glutamine. In mature somatic embryos GS and NR were active. NR activity was highest, if embryos were matured on Gln + NO3 and notably lower if matured on Gln or NH4NO3. The tendentially highest GS activity was found if embryos were generated on NH4NO3. A striking change in nitrogen metabolism was the steady increase in GS activity from not detectable at proliferation stage, through easily detectable during maturation up to high activity in SE plantlets.

  • 135. Davidson, Iain F.
    et al.
    Goetz, Daniela
    Zaczek, Maciej P.
    Molodtsov, Maxim I.
    in't Veld, Pim J. Huis
    Weissmann, Florian
    Litos, Gabriele
    Cisneros, David A.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Research Institute of Molecular Pathology (IMP), Vienna, Austria.
    Ocampo-Hafalla, Maria
    Ladurner, Rene
    Uhlmann, Frank
    Vaziri, Alipasha
    Peters, Jan-Michael
    Rapid movement and transcriptional re-localization of human cohesin on DNA2016In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 24, p. 2671-2685Article in journal (Refereed)
    Abstract [en]

    The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

  • 136. de Albuquerque Wanderley, Maria Carolina
    et al.
    Martín, Carlos
    Department of Chemistry and Chemical Engineering, University of Matanzas, Matanzas, Cuba; vTI-Institute for Wood Technology and Wood Biology, Hamburg, Germany.
    de Moraes Rocha, George Jackson
    Gouveia, Ester Ribeiro
    Increase in ethanol production from sugarcane bagasse based on combined pretreatments and fed-batch enzymatic hydrolysis2013In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 128, p. 448-453Article in journal (Refereed)
    Abstract [en]

    Enzymatic hydrolysis of pretreated sugarcane bagasse was performed to investigate the production of ethanol. The sugarcane bagasse was pretreated in a process combining steam explosion and alkaline delignification. The lignin content decreased to 83%. Fed-batch enzymatic hydrolyses was initiated with 8% (w/v) solids loading, and 10 FPU/g cellulose. Then, 1% solids were fed at 12, 24 or 48 h intervals. After 120 h, the hydrolysates were fermented with Saccharomyces cerevisiae UFPEDA 1238, and a fourfold increase in ethanol production was reached when fed-batch hydrolysis with a 12-h addition period was used for the steam pretreated and delignified bagasse.

  • 137.
    De Bleser, Helena
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Crosstalk of ethylene and gibberellins during wood formation in hybridaspen2013Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Both gibberellins (GAs) and ethylene (ET) or its in planta precursor 1‐Aminocyclopropane-1‐carboxylic acid (ACC) stimulate cambial cell division and modify wood development when exogenously applied to wood forming tissues of trees. Furthermore both hormones are involved in tension wood (TW) formation in leaned trees. In Arabidopsis a cross‐talk of ET and GA on a molecular level has been demonstrated. We have examined here the effects of GA and ACC, alone and in combination, on wood development in hybrid aspen (Populus tremula x tremuloides) to investigate their potential of cross-talk during wood development. The response of selected transcripts involved in GA, ET and auxin signaling, biosynthesis and transport was inspected in the total stem of T89 trees after 10 hours of treatment with quantitative real‐time PCR (qPCR). Analysis of the phenotype, anatomy and chemistry of wild-type, ethylene-insensitive and GA‐deficient trees after 2 weeks of treatment emphasized that a cross‐talk between GA and ACC is plausible. Based on primary growth characteristics, GA and ACC seemed to be partially redundant. Lignin stainings suggest antagonistic interactions, while fiber to vessel ratios and the distribution of G‐layers put forward a collaborating action. Diffuse reflectance FT‐IR demonstrates that functional GA and ACC signalling are needed to induce differences in chemical composition.

     

  • 138.
    de La Torre, Amanda R.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Plant Sciences, University of California–Davis, Davis, CA.
    Li, Zhen
    Van de Peer, Yves
    Ingvarsson, Pär K.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Plant Biology, Uppsala Biocenter, Swedish University of Agr icultural Sciences, Uppsala, Sweden.
    Contrasting Rates of Molecular Evolution and Patterns of Selection among Gymnosperms and Flowering Plants2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 6, p. 1363-1377Article in journal (Refereed)
    Abstract [en]

    The majority of variation in rates of molecular evolution among seed plants remains both unexplored and unexplained. Although some attention has been given to flowering plants, reports of molecular evolutionary rates for their sister plant clade (gymnosperms) are scarce, and to our knowledge differences in molecular evolution among seed plant clades have never been tested in a phylogenetic framework. Angiosperms and gymnosperms differ in a number of features, of which contrasting reproductive biology, life spans, and population sizes are the most prominent. The highly conserved morphology of gymnosperms evidenced by similarity of extant species to fossil records and the high levels of macrosynteny at the genomic level have led scientists to believe that gymnosperms are slow-evolving plants, although some studies have offered contradictory results. Here, we used 31,968 nucleotide sites obtained from orthologous genes across a wide taxonomic sampling that includes representatives of most conifers, cycads, ginkgo, and many angiosperms with a sequenced genome. Our results suggest that angiosperms and gymnosperms differ considerably in their rates of molecular evolution per unit time, with gymnosperm rates being, on average, seven times lower than angiosperm species. Longer generation times and larger genome sizes are some of the factors explaining the slow rates of molecular evolution found in gymnosperms. In contrast to their slow rates of molecular evolution, gymnosperms possess higher substitution rate ratios than angiosperm taxa. Finally, our study suggests stronger and more efficient purifying and diversifying selection in gymnosperm than in angiosperm species, probably in relation to larger effective population sizes.

  • 139.
    De La Torre, Amanda R.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Univ British Columbia, Dept Forest & Conservat Sci, Ctr Forest Conservat Genet, Vancouver, BC V6T 1Z4, Canada.
    Roberts, David R.
    Aitken, Sally N.
    Genome-wide admixture and ecological niche modelling reveal the maintenance of species boundaries despite long history of interspecific gene flow2014In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 23, no 8, p. 2046-2059Article in journal (Refereed)
    Abstract [en]

    The maintenance of species boundaries despite interspecific gene flow has been a continuous source of interest in evolutionary biology. Many hybridizing species have porous genomes with regions impermeable to introgression, conferring reproductive barriers between species. We used ecological niche modelling to study the glacial and postglacial recolonization patterns between the widely hybridizing spruce species Picea glauca and P.engelmannii in western North America. Genome-wide estimates of admixture based on a panel of 311 candidate gene single nucleotide polymorphisms (SNP) from 290 genes were used to assess levels of admixture and introgression and to identify loci putatively involved in adaptive differences or reproductive barriers between species. Our palaeoclimatic modelling suggests that these two closely related species have a long history of hybridization and introgression, dating to at least 21000years ago, yet species integrity is maintained by a combination of strong environmental selection and reduced current interspecific gene flow. Twenty loci showed evidence of divergent selection, including six loci that were both F-st outliers and associated with climatic gradients, and fourteen loci that were either outliers or showed associations with climate. These included genes responsible for carbohydrate metabolism, signal transduction and transcription factors.

  • 140.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Kleczkowski, Leszek A.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Substrate specificity and inhibitor sensitivity of plant UDP-sugar producing pyrophosphorylasesManuscript (preprint) (Other academic)
  • 141.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Meng, Meng
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Gornicka, Agnieszka
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase2012In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 79, p. 39-45Article in journal (Refereed)
    Abstract [en]

    UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

  • 142.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Öberg, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kleczkowski, Leszek A.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 6, p. 1093-1107Article in journal (Other academic)
    Abstract [en]

    UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

  • 143.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Öberg, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kleczkowski, Leszek A.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases2018In: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 13, no 8, article id e1507406Article in journal (Refereed)
    Abstract [en]

    UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

  • 144. Deem, A
    et al.
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blackgrove, T
    Vayl, A
    Coffey, B
    Mathur, R
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Malkova, A
    Department of Biology, School of Science, IUPUI, Indianapolis, Indiana, United States of America.
    Break-induced replication is highly inaccurate2011In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885Article in journal (Refereed)
    Abstract [en]

    DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR) is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs) rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.

  • 145. Dejonghe, Wim
    et al.
    Kuenen, Sabine
    Mylle, Evelien
    Vasileva, Mina
    Keech, Olivier
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Viotti, Corrado
    Swerts, Jef
    Fendrych, Matyas
    Ortiz-Morea, Fausto Andres
    Mishev, Kiril
    Delang, Simon
    Scholl, Stefan
    Zarza, Xavier
    Heilmann, Mareike
    Kourelis, Jiorgos
    Kasprowicz, Jaroslaw
    Nguyen, Le Son Long
    Drozdzecki, Andrzej
    Van Houtte, Isabelle
    Szatmari, Anna-Maria
    Majda, Mateusz
    Baisa, Gary
    Bednarek, Sebastian York
    Robert, Stephanie
    Audenaert, Dominique
    Testerink, Christa
    Munnik, Teun
    Van Damme, Daniel
    Heilmann, Ingo
    Schumacher, Karin
    Winne, Johan
    Friml, Jiri
    Verstreken, Patrik
    Russinova, Eugenia
    Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 11710Article in journal (Refereed)
    Abstract [en]

    ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

  • 146.
    Del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmfeldt, Linda
    Togneri, Peter
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A hyper-mutant of the unusual σ70-Pr promoter bypasses synergistic ppGpp/DksA co-stimulation2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 14, p. 5853-5865Article in journal (Refereed)
    Abstract [en]

    The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ70-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the −11 position of the extremely suboptimal −10 element of Pr that underlies both poor binding of σ70-RNAP and a slow rate of open complex formation—the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation.

  • 147.
    del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Inter-sigmulon communication through topological promoter coupling2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 20, p. 9638-9649Article in journal (Refereed)
    Abstract [en]

    Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative sigma-factors. This is the case for the non-overlapping sigma(70)- and sigma(54)-dependent promoters that control production of the substrate-responsive regulator and enzymes for (methyl) phenol catabolism. Here, using an array of in vivo and in vitro assays, we identify transcription-driven supercoiling arising from the sigma(54)-promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the sigma(70)-promoter. The non-overlapping 'back-to-back' configuration of a powerful sigma(54)-promoter and weak sigma(70)-promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the sigma(70)-promoter subservient to signals that elicit sigma(54)-dependent transcription without it possessing a cognate binding site for the sigma(54)-RNA polymerase holoenzyme. This mode of control has the potential to be a prevalent, but hitherto unappreciated, mechanism by which bacteria adjust promoter activity to gain appropriate transcriptional control.

  • 148. Dellino, Gaetano I
    et al.
    Schwartz, Yuri B
    University of Geneva, Switzerlandc.
    Farkas, Gabriella
    McCabe, Donna
    Elgin, Sarah C R
    Pirrotta, Vincenzo
    Polycomb silencing blocks transcription initiation.2004In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 13, no 6, p. 887-93Article in journal (Refereed)
    Abstract [en]

    Polycomb (PcG) complexes maintain the silent state of target genes. The mechanism of silencing is not known but has been inferred to involve chromatin packaging to block the access of transcription factors. We have studied the effect of PcG silencing on the hsp26 heat shock promoter. While silencing does decrease the accessibility of some restriction enzyme sites to some extent, it does not prevent the binding of TBP, RNA polymerase, or the heat shock factor to the hsp26 promoter, as shown by chromatin immunoprecipitation. However, we find that in the repressed state, the RNA polymerase cannot initiate transcription. We conclude that, rather than altering chromatin structure to block accessibility, PcG silencing in this construct targets directly the activity of the transcriptional machinery at the promoter.

  • 149.
    Demakov, Sergei
    et al.
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Gortchakov, Andrei
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Schwartz, Yuri B
    Institute of Cytology & Genetics, Russian Academy of Sciences, Russia.
    Semeshin, Valery
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Campuzano, Sonsoles
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Modolell, Juan
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Zhimulev, Igor
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes: evidence for two types of interband regions2004In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 122, no 3, p. 311-324Article in journal (Refereed)
    Abstract [en]

    The 3A and 60E regions of Drosophila melanogaster polytene chromosomes containing inserted copies of the P[1ArB] transposon have been subjected to an electron microscopic (EM) analysis. We show that both inserts led to formation of new bands within the interband regions 3A4/A6 and 60E8-9/E10. This allowed us to clone DNA of these interbands. Their sequences, as well as those of DNA from other four interbands described earlier, have been analyzed. We have found that, with the exception of 60E8-9/E10 interband, all other five regions under study corresponded to 5' or 3' ends of genes. We have further obtained the evidence for 60E8-9/E10 interband to harbor the 'housekeeping' RpL19 gene, which is transcribed in many tissues, including salivary glands. Based upon the genetic heterogeneity of the interbands observed a revised model of polytene chromosome organization is discussed.

  • 150.
    den Hollander, Jürgen
    et al.
    III. Medical Department, Technische Universität München, Munich, Germany.
    Rimpi, Sara
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Doherty, Joanne R
    Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
    Rudelius, Martina
    Department of Pathology, Technische Universität München, Munich, Germany.
    Buck, Andreas
    Department of Nuclear Medicine, Technische Universität München, Munich, Germany.
    Kremer, Marcus
    Department of Pathology, Technische Universität München, Munich, Germany.
    Graf, Nikolas
    III. Medical Department, Technische Universität München, Munich, Germany.
    Scheerer, Markus
    III. Medical Department, Technische Universität München, Munich, Germany.
    Hall, Mark
    Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
    von Bubnoff, Nikolas
    III. Medical Department, Technische Universität München, Munich, Germany.
    Duyster, Justus
    III. Medical Department, Technische Universität München, Munich, Germany.
    Peschel, Christian
    III. Medical Department, Technische Universität München, Munich, Germany.
    Cleveland, John L
    Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
    Nilsson, Jonas A
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Keller, Ulrich
    III. Medical Department, Technische Universität München, Munich, Germany.
    Aurora kinases A and B are Myc targets essential for maintenance of the malignant stateManuscript (preprint) (Other academic)
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