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  • 1.
    Abbott, Jessica K.
    et al.
    Queen's University.
    Bensch, S.
    Lund University.
    Gosden, Thomas P.
    Lund University.
    Svensson, Erik I.
    Lund University.
    Patterns of differentiation in a colour polymorphism and in neutral markers reveal rapid genetic changes in natural damselfly populations2008Inngår i: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 17, nr 6, s. 1597-1604Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The existence and mode of selection operating on heritable adaptive traits can be inferred by comparing population differentiation in neutral genetic variation between populations (often using F(ST) values) with the corresponding estimates for adaptive traits. Such comparisons indicate if selection acts in a diversifying way between populations, in which case differentiation in selected traits is expected to exceed differentiation in neutral markers [F(ST )(selected) > F(ST )(neutral)], or if negative frequency-dependent selection maintains genetic polymorphisms and pulls populations towards a common stable equilibrium [F(ST) (selected) < F(ST) (neutral)]. Here, we compared F(ST) values for putatively neutral data (obtained using amplified fragment length polymorphism) with estimates of differentiation in morph frequencies in the colour-polymorphic damselfly Ischnura elegans. We found that in the first year (2000), population differentiation in morph frequencies was significantly greater than differentiation in neutral loci, while in 2002 (only 2 years and 2 generations later), population differentiation in morph frequencies had decreased to a level significantly lower than differentiation in neutral loci. Genetic drift as an explanation for population differentiation in morph frequencies could thus be rejected in both years. These results indicate that the type and/or strength of selection on morph frequencies in this system can change substantially between years. We suggest that an approach to a common equilibrium morph frequency across all populations, driven by negative frequency-dependent selection, is the cause of these temporal changes. We conclude that inferences about selection obtained by comparing F(ST) values from neutral and adaptive genetic variation are most useful when spatial and temporal data are available from several populations and time points and when such information is combined with other ecological sources of data.

  • 2.
    Abdurakhmanov, Eldar
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Discovery and evaluation of direct acting antivirals against hepatitis C virus2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued.

    In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants.

    By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3.

    In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV.

    An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate.

    Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.

    Delarbeid
    1. Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
    Åpne denne publikasjonen i ny fane eller vindu >>Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
    Vise andre…
    2016 (engelsk)Inngår i: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, nr 12, s. 2603-2620Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

    Emneord
    Hepatitis C virus; Drug resistance; Pyrazinone; NS3 protease inhibitors; R155K
    HSV kategori
    Forskningsprogram
    Läkemedelskemi
    Identifikatorer
    urn:nbn:se:uu:diva-243315 (URN)10.1016/j.bmc.2016.03.066 (DOI)000376727800002 ()27160057 (PubMedID)
    Forskningsfinansiär
    Swedish Research Council, D0571301
    Tilgjengelig fra: 2015-02-08 Laget: 2015-02-08 Sist oppdatert: 2017-12-04bibliografisk kontrollert
    2. Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
    Åpne denne publikasjonen i ny fane eller vindu >>Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-265295 (URN)
    Tilgjengelig fra: 2015-10-26 Laget: 2015-10-26 Sist oppdatert: 2016-01-13
    3. Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
    Åpne denne publikasjonen i ny fane eller vindu >>Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
    Vise andre…
    2013 (engelsk)Inngår i: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 97, nr 3, s. 356-368Artikkel i tidsskrift (Annet vitenskapelig) Published
    Abstract [en]

    Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

    Emneord
    HCV, NS5B, filibuvir, VX-222, tegobuvir, allosteric inhibitor, induced fit, kinetics, chemodynamics, thermodynamics
    HSV kategori
    Forskningsprogram
    Biokemi; Biokemi
    Identifikatorer
    urn:nbn:se:uu:diva-171996 (URN)10.1016/j.antiviral.2012.12.027 (DOI)000317709400018 ()
    Tilgjengelig fra: 2012-04-03 Laget: 2012-03-31 Sist oppdatert: 2017-12-07bibliografisk kontrollert
    4. Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    Åpne denne publikasjonen i ny fane eller vindu >>Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-265287 (URN)
    Tilgjengelig fra: 2015-10-26 Laget: 2015-10-26 Sist oppdatert: 2016-01-13
    5. A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    Åpne denne publikasjonen i ny fane eller vindu >>A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-265290 (URN)
    Tilgjengelig fra: 2015-10-26 Laget: 2015-10-26 Sist oppdatert: 2016-01-13
    6. Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Åpne denne publikasjonen i ny fane eller vindu >>Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-265292 (URN)
    Tilgjengelig fra: 2015-10-26 Laget: 2015-10-26 Sist oppdatert: 2016-01-13
  • 3. Abdurakhmanov, Eldar
    et al.
    Danielson, Helena
    A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discoveryManuskript (preprint) (Annet vitenskapelig)
  • 4.
    Abdurakhmanov, Eldar
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Solbak, Sara
    Danielson, Helena
    Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative studyManuskript (preprint) (Annet vitenskapelig)
  • 5.
    Abdurakhmanov, Eldar
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Solbak, Sara Oie
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Danielson, U. Helena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase2017Inngår i: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 9, nr 6, artikkel-id 151Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

  • 6. Abel, John H.
    et al.
    Drawert, Brian
    Hellander, Andreas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Institutionen för informationsteknologi, Avdelningen för beräkningsvetenskap. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Institutionen för informationsteknologi, Tillämpad beräkningsvetenskap.
    Petzold, Linda R.
    GillesPy: A Python package for stochastic model building and simulation2016Inngår i: IEEE Life Sciences Letters, E-ISSN 2332-7685, Vol. 2, s. 35-38Artikkel i tidsskrift (Fagfellevurdert)
  • 7.
    Aboye, Teshome L.
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    Strömstedt, Adam A.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    Gunasekera, Sunithi
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    Bruhn, Jan G.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    El-Seedi, Hesham
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    Rosengren, K. Johan
    Göransson, Ulf
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    A Cactus-Derived Toxin-Like Cystine Knot Peptide with Selective Antimicrobial Activity2015Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, nr 7, s. 1068-1077Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide-based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three-dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep-AMP1 from Echinopsis pachanoi. The structure of Ep-AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec-AMP1 in the plant C6-type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.

  • 8.
    Abramson, Jeff
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
    Structural studies on the integral membrane protein, ubiquinol oxidase from Escherichia coli2001Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Heme-copper oxidases are redox-driven proton pumps that couple the reduction of molecular oxygen to water with the vectorial translocation of protons across the membrane. The proton gradient generated by heme-copper oxidases and the other members of the aerobic respiratory chain is ultimately used to drive the synthesis of ATP. There are two main branches of the heme-copper oxidases that are characterized by the electron donating substrate; the cytochrome c oxidases, which use cytochrome c as the electron donor, and the ubiquinol oxidases, which use a lipid-soluble molecule, ubiquinol, as their electron donor. These enzymes share important structural and functional features.

    This thesis presents the procedures that have led to the first crystal structure of a ubiquinol oxidase, cytochrome bo, oxidase from Escherichia coli, at a resolution of 3.5 Å. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however the membrane spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer. No such structural feature is present in cytochrome c oxidases. Mutagenesis studies on residues in this region strongly suggest that this area forms a ubiquinone binding site. A comparison of this region with known ubiquinone binding sites shows remarkable similarities. In light of these findings specific roles for these polar residues is proposed in electron and proton transfer in ubiquinol oxidase.

    A fusion protein of cytochrome bo3-Protein Z was generated in an attempt to increase the hydrophilic surface of the protein, thus extending protein-protein contacts within the crystal lattice structure. Such an approach can be used to facilitate crystallization.

  • 9.
    Adler, Marlen
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli2014Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia.

    Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function.

    In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

    Delarbeid
    1. Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli
    Åpne denne publikasjonen i ny fane eller vindu >>Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli
    2013 (engelsk)Inngår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, nr 1, s. 51-59Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Objectives: To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Methods: Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results: Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Conclusions: Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

    Emneord
    Fitness cost, Gene amplification, Mutations, Plasmids
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-192026 (URN)10.1093/jac/dks368 (DOI)000312646300010 ()
    Tilgjengelig fra: 2013-01-24 Laget: 2013-01-15 Sist oppdatert: 2017-12-06bibliografisk kontrollert
    2. Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model
    Åpne denne publikasjonen i ny fane eller vindu >>Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model
    Vise andre…
    2013 (engelsk)Inngår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, nr 6, s. 1319-1326Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    OBJECTIVES:

    Ertapenem resistance is increasing in Enterobacteriaceae. The production of extended-spectrum β-lactamases (ESBLs) and reduced expression of outer membrane porins are major mechanisms of resistance in ertapenem-resistant Klebsiella pneumoniae. Less is known of ertapenem resistance in Escherichia coli. The aim of this study was to explore the impact of ESBL production in E. coli on the antibacterial activity of ertapenem.

    METHODS:

    Two E. coli strains, with and without ESBL production, were exposed to ertapenem in vitro for 48 h at concentrations simulating human pharmacokinetics with conventional and higher dosages.

    RESULTS:

    Isolates with non-susceptibility to ertapenem (MICs 0.75-1.5 mg/L) were detected after five of nine time-kill experiments with the ESBL-producing strain. All of these isolates had ompR mutations, which reduce the expression of outer membrane porins OmpF and OmpC. Higher dosage did not prevent selection of porin-deficient subpopulations. No mutants were detected after experiments with the non-ESBL-producing strain. Compared with other experiments, experiments with ompR mutants detected in endpoint samples showed significantly less bacterial killing after the second dose of ertapenem. Impaired antibacterial activity against E. coli with ESBL production and ompR mutation was also demonstrated in time-kill experiments with static antibiotic concentrations.

    CONCLUSIONS:

    The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-197878 (URN)10.1093/jac/dkt044 (DOI)000319468900016 ()23478794 (PubMedID)
    Tilgjengelig fra: 2013-04-05 Laget: 2013-04-05 Sist oppdatert: 2017-12-06bibliografisk kontrollert
    3. Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
    Åpne denne publikasjonen i ny fane eller vindu >>Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
    2016 (engelsk)Inngår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, nr 5, s. 1188-1198Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-221428 (URN)10.1093/jac/dkv475 (DOI)000376291300008 ()26869688 (PubMedID)
    Forskningsfinansiär
    Swedish Research Council Formas, 2013-5476-25194-9EU, European Research Council, 282004
    Tilgjengelig fra: 2014-03-31 Laget: 2014-03-31 Sist oppdatert: 2017-12-05bibliografisk kontrollert
    4. High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms
    Åpne denne publikasjonen i ny fane eller vindu >>High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms
    Vise andre…
    2014 (engelsk)Inngår i: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, nr 6, s. 1526-1535Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-221431 (URN)10.1093/molbev/msu111 (DOI)000337067400019 ()
    Tilgjengelig fra: 2014-03-31 Laget: 2014-03-31 Sist oppdatert: 2017-12-05bibliografisk kontrollert
  • 10.
    Adler, Marlen
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Anjum, Mehreen
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Andersson, Dan I.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sandegren, Linus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli2016Inngår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, nr 5, s. 1188-1198Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

  • 11.
    Adler, Marlen
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Anjum, Mehreen
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Berg, Otto, G.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Beräknings- och systembiologi.
    Andersson, Dan I.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sandegren, Linus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms2014Inngår i: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, nr 6, s. 1526-1535Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

  • 12.
    Afshari Kashanian, Elisa
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Detection of celery (Apium graveolens) in food with Real-Time PCR2006Independent thesis Basic level (degree of Bachelor), 10 poäng / 15 hpOppgave
    Abstract [en]

    Directive EC 2003/89/EC of the European Parliament and of the Council states that certain

    ingredients and products derived there of known to cause allergen reactions must always be

    declared. Furthermore labelling is mandatory irrespective of the amount included. The National

    Food Administration therefore needs methods for monitoring the presence of allergens in food.

    Methods already exist for most of the allergens on the EU-list, but an operational method for

    celery (Apium graveolens) is missing.

    A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery

    mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation

    of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be

    specific for celery, producing a 113 bp fragment with two celery varieties and negative results

    with other closely selected species commonly present together with celery in food products (12

    samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome

    copies. When evaluated with model samples of celery in meat, a detection limit of less than

    0,01 % was determined. When used to analyse food products from the market, six out of seven

    products declared to contain celery were correctly identified as positive.

  • 13.
    Ahlström, Anna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Testing the specificity of the pBAD arabinose reporter2017Independent thesis Basic level (degree of Bachelor), 10 poäng / 15 hpOppgave
    Abstract [en]

    The project highlights Salmonella enterica subspecies enterica serovar Typhimurium (S. Tm)'s ability to metabolize simple sugars released from dead commensal bacteria, by using the pBAD (araBAD promoter) system as a reporter of L-arabinose availability. Using bioinformatics and homology of conserved L-arabinose transporter genes shared in Escherichia coli K12 (E. coli) and S. Tm, we aimed to create a S. Tm mutant strain unable to obtain L-arabinose from it environment. During the projects course of time it was discovered that L-arabinose transporters are not a shared gene trait between E. coli and S. Tm, and that putative L-arabinose transporter orthologues may exists in the S. Tm genome.

  • 14.
    Ahlsén, Göran
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
    Structure-activity and resistance studies of HIV-1 protease inhibitors2000Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The present investigation was undertaken in order to identify inhibitors of HIV-1 protease that would be efficient in vivo and against HIV-1 protease carrying mutations known to confer resistance to inhibitors in clinical use. A second interest was to understand details of inhibitory mechanisms and to gain understanding of the molecular details of resistance.

    Linear inhibitors of transition-state type showed to have a resistance pattern similar to protease inhibitors in clinical use, whereas cyclic inhibitors of sulfonamide were somewhat different in their inhibitory profiles. It was found that mutation L90M in some situations could lessen the decrease in overall efficiency suffered by the enzyme when aquiring other mutations. Also presented are results from the characterization of double mutation I84V/L90M, formerly not investigated. Testing of triple and quadruple mutant confirmed the additive features of some mutations. In an attempt to find new leads for inhibitor development, extracts from bee propolis, a natural product, was investigated, and it was found that one extract inhibited wild-type enzyme with an I50-value of 0.2 μg/mL. Even more interesting is the result that propolis extract also inhibited all the investigated mutant enzymes.

  • 15.
    Ahmad, Shabbir
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC.
    Hysteretic Behavior, Regioselectivity, and Role of Salt Bridging Residues at the Domain Interface of Potato Epoxide Hydrolase StEH1, Site-Directed Mutagenesis and Kinetic Study2009Independent thesis Advanced level (degree of Master (Two Years)), 30 poäng / 45 hpOppgave
  • 16.
    Ahmadova, Nigar
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
    Mamedov, Fikret
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
    Formation of tyrosine radicals in photosystem II under far-red illumination2018Inngår i: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 136, nr 1, s. 93-106Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Photosystem II (PS II) contains two redox-active tyrosine residues on the donor side at symmetrical positions to the primary donor, P680. TyrZ, part of the water-oxidizing complex, is a preferential fast electron donor while TyrD is a slow auxiliary donor to P680 +. We used PS II membranes from spinach which were depleted of the water oxidation complex (Mn-depleted PS II) to study electron donation from both tyrosines by time-resolved EPR spectroscopy under visible and far-red continuous light and laser flash illumination. Our results show that under both illumination regimes, oxidation of TyrD occurs via equilibrium with TyrZ at pH 4.7 and 6.3. At pH 8.5 direct TyrD oxidation by P680 + occurs in the majority of the PS II centers. Under continuous far-red light illumination these reactions were less effective but still possible. Different photochemical steps were considered to explain the far-red light-induced electron donation from tyrosines and localization of the primary electron hole (P680 +) on the ChlD1 in Mn-depleted PS II after the far-red light-induced charge separation at room temperature is suggested.

  • 17. Ahmed, Aisha S
    et al.
    Li, Jian
    Erlandsson-Harris, Helena
    Stark, André
    Bakalkin, Georgy
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaceutisk biovetenskap.
    Mahmood, Ahmed
    Suppression of pain and joint destruction by inhibition of the proteasome system in experimental osteoarthritis2012Inngår i: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 153, nr 1, s. 18-26Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Osteoarthritis is a degenerative joint disease with pain and loss of joint function as major pathological features. Recent studies show that proteasome inhibitors reduce pain in various pathological conditions. We evaluated the effects of MG132, a reversible proteasome inhibitor on pain and joint destruction in a rat model of osteoarthritis. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate into the rat knee. Knee joint stiffness was scored and nociception was evaluated by mechanical pressure applied to the respective hind paw. Knee joint destruction was assessed by radiological and histological analyses. Expression of matrix metalloproteinase-3 (MMP-3) was analyzed by quantitative reverse transcription polymerase chain reaction in the knee articular cartilage. Expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the dorsal root ganglia (L4–L6) by quantitative reverse transcription polymerase chain reaction and in the knee joints by immunohistochemistry. Our results indicate that daily treatment of osteoarthritic rats with MG132 significantly increases their mobility while the swelling, pain thresholds, and pathological features of the affected joints were reduced. Furthermore, the upregulated expression of MMP-3, SP, and CGRP in the arthritic rats was normalized by MG132 administration. We conclude that the proteasome inhibitor MG132 reduces pain and joint destruction, probably by involving the peripheral nervous system, and that changes in SP and CGRP expression correlate with alterations in behavioural responses. Our findings suggest that nontoxic proteasome inhibitors may represent a novel pharmacotherapy for osteoarthritis.

  • 18.
    Ahnoff, Martin
    et al.
    Univ Gothenburg, Dept Chem Mol Biol, SE-41296 Gothenburg, Sweden.;Denator AB, Gothenburg, Sweden..
    Cazares, Lisa H.
    US Army Med Res Inst Infect Dis, Mol & Translat Sci, Frederick, MD 21702 USA.;US Army Med Res & Mat Command, DoD Biotechnol High Performance Comp Software App, Telemed & Adv Technol Res Ctr, Ft Detrick, MD 21702 USA..
    Sköld, Karl
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin. Denator AB, Gothenburg, Sweden.;Uppsala Univ, Dept Med Sci Canc Pharmacol & Computat Med, Uppsala, Sweden..
    Thermal inactivation of enzymes and pathogens in biosamples for MS analysis2015Inngår i: Bioanalysis, ISSN 1757-6180, E-ISSN 1757-6199, Vol. 7, nr 15, s. 1885-1899Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (approximate to 1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

  • 19. Aili, Daniel
    et al.
    Enander, Karin
    Baltzer, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Kemiska institutionen.
    Liedberg, Bo
    Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing2007Inngår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, nr 3, s. 532-534Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    This contribution describes how de novo designed synthetic helix–loop–helix polypeptides are utilized tocontrol the assembly of gold nanoparticles and as scaffolds for biosensing. The synthetic polypeptides aredesigned to fold into a four-helix bundle upon dimerization. When immobilized on gold nanoparticles,dimerization and folding occur between peptides on neighbouring particles as an effect of particleaggregation and the folded polypeptides are rigid enough to keep the particles separated at a distancecorresponding to the size of the four-helix bundle. Moreover, peptide dimerization offers a convenientroute to assemble nanoparticles into hybrid multilayers on planar substrates. The drastic change in theresonance conditions of the localized nanoparticle surface plasmon upon particle aggregation is shown tobe useful for optical detection of biomolecular interactions.

  • 20.
    Aksoy, N. H.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi. Aksaray Univ, Dept Biochem, Aksaray, Turkey..
    Mannervik, B.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Inhibitory effects of ethacrynic acid on glutathione S-transferase A1-1 from Callithrix jacchus2015Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, s. 348-348Artikkel i tidsskrift (Annet vitenskapelig)
  • 21.
    Alderborn, Anders
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för genetik och patologi.
    Sundström, Jens
    Soeria-Atmadja, Daniel
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Sandberg, Martin
    Andersson, H. Christer
    Hammerling, Ulf
    Genetically modified plants for non-food or non-feed purposes: straightforward screening for their appearance in food and feed2010Inngår i: Food and Chemical Toxicology, ISSN 0278-6915, E-ISSN 1873-6351, Vol. 48, nr 2, s. 453-464Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Genetically modified (GM) plants aimed at producing food/feed are part of regular agriculture in many areas of the World. Commodity plants have also found application as bioreactors, designated non-food/non-feed GM (NFGM) plants, thereby making raw material for further refinement to industrial, diagnostic or pharmaceutical preparations. Many among them may pose health challenge to consumers or livestock animals, if occurring in food/feed. NFGM plants are typically released into the environment, but are grown under special oversight and any among several containment practices, none of which provide full protection against accidental dispersal. Adventitious admixture with food or feed can occur either through distributional mismanagement or as a consequence of gene flow to plant relatives. To facilitate NFGM surveillance we propose a new mandatory tagging of essentially all such plants, prior to cultivation or marketing in the European Union. The suggested tag--Plant-Made Industrial or Pharmaceutical Products Tag (PMIP-T)--is envisaged to occur as a transgenic silent DNA identifier in host plants and designed to enable technically simple identification and characterisation of any NFGM. Implementation of PMIP-T would permit inexpensive, reliable and high-throughput screening for NFGM specifically. The paper outlines key NFGM prospects and challenges as well as the PMIP-T concept.

  • 22.
    Alexis, Laura
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Alftrén, Ida
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Elvmarker, Josefin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Mach, Klara-Maria
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    Nyman, Rebecca
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning.
    DETEKTERA MERA!: - En studie av LigandTracer® och andra vanliga detektionstekniker för interaktion inom antikroppsutveckling2013Independent thesis Basic level (degree of Bachelor), 10 poäng / 15 hpOppgave
    Abstract [sv]

    Ridgeview Instruments AB är ett företag, med säte i Uppsala, som bland annat utvecklar mjukvara för detektionsanalys och detektionsinstrumenten LigandTracer®. Instrumenten detekterar cellinteraktioner i realtid och tillhandahåller information om hur interaktion mellan undersökt ligand och receptor sker. LigandTracer® används bland annat vid cancerstudier och affinitetsstudier av antikroppsinbindning till antigener på celler.

     

    LigandTracer® och teknologin bakom den har studerats för att hitta de främsta styrkorna samt utvecklingsområden i jämförelse med andra detektionstekniker. Dessa tekniker är surface plasmon resonance, BioLayer Interferometry, quartz crystal microbalance samt surface acoustic wave. Främsta styrkan hos detektion med LigandTracer® är att stor mängd data som beskriver interaktionerna erhålls. Information av denna typ är viktig för att få en helhetsbild av cellers ytprotein. Därför kan instrumenten exempelvis användas som kvalitetssäkrande steg i antikroppsprocessen. LigandTracer® kan även användas för studier av hela celler och vävnader. Utöver detta finns styrkor som minimerar det laborativa arbetet, så som tvättar i prepareringssteg. Detta gör att både tid och reagens sparas.

     

    En utvecklingsmöjlighet för LigandTracer® är egenskapen att kunna detektera fler antikroppar och cellinjer samtidigt. Då mjukvaran TraceDrawer™ är ett viktigt tillbehör för utvärderingen av detektionen, skulle utveckling med avseende på celldynamiken och receptorernas rörelsemönster kunna förbättra instrumenten ytterligare.

  • 23.
    Alfredsson Timmins, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Functional organisation of the cell nucleus in the fission yeast, Schizosaccharomyces pombe2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In eukaryotes the genome adopts a non-random spatial organisation, which is important for gene regulation. However, very little is known about the driving forces behind nuclear organisation. In the simple model eukaryote fission yeast, Schizosaccharomyces pombe, it has been known for a long time that transcriptionally repressed heterochromatin localise to the nuclear membrane (NM); the centromeres attaches to spindle pole body (SPB), while the telomeres are positioned at the NM on the opposite side of the nucleus compared to the SPB. Studies presented in this thesis aimed at advancing our knowledge of nuclear organisation in Schizosaccharomyces pombe.

    We show that the heterochromatic mating-type region localises to the NM in the vicinity of the SPB. This positioning was completely dependent on Clr4, a histone methyl transferase crucial for the formation of heterochromatin. Additional factors important for localisation were also identified: the chromo domain protein Swi6, and the two boundary elements IR-L and IR-R surrounding this locus. We further identify two other chromo domain proteins; Chp1 and Chp2, as crucial factors for correct subnuclear localisation of this region. From these results we suggest that the boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin.

    Gene regulation can affect the subnuclear localisation of genes. Using nitrogen starvation in S. pombe as a model for gene induction we determined the subnuclear localisation of two gene clusters repressed by nitrogen: Chr1 and Tel1. When repressed these loci localise to the NM, and this positioning is dependent on the histone deacetylase Clr3. During induction the gene clusters moved towards the nuclear interior in a transcription dependent manner.

    The knowledge gained from work presented in this thesis, regarding nuclear organisation in the S. pombe model system, can hopefully aid to a better understanding of human nuclear organisation.

    Delarbeid
    1. The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast
    Åpne denne publikasjonen i ny fane eller vindu >>The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast
    2007 (engelsk)Inngår i: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 120, nr 11, s. 1935-1943Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The genome has a non-random spatial distribution in the cell nucleus. In Schizosaccharomyces pombe, it has been shown that the centromeres, telomeres and the mating-type region localize to the nuclear membrane (NM), the former by attaching to the spindle pole body (SPB). In addition, reporter genes inserted into these areas are transcriptionally repressed due to the formation of specialized chromatin structures. Performing live cell analysis we found that in a wild-type strain the mating-type region was positioned in the proximity of the SPB, the location where the pericentromeric heterochromatin is also found. In a strain lacking the histone methyltransferase, Clr4, crucial for the formation of heterochromatin, the mating-type region had a random localization in the nucleus. Moreover, in a strain where the two boundary elements IR-L and IR-R had been deleted the mating-type region was displaced from its position at the proximity of the SPB, but remained in the vicinity of the NM. Moreover, in all investigated strains with silencing deficiencies the distance between the mating-type region and the SPB increased. This result indicates a correlation between transcriptional derepression and displacement of the region. Two different models of how the mating-type chromatin is organized in the nucleus are discussed.

    Emneord
    fission yeast, heterochromatin, silencing, subnuclear localization
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-13023 (URN)10.1242/jcs.03457 (DOI)000246665300013 ()17504808 (PubMedID)
    Tilgjengelig fra: 2009-08-05 Laget: 2009-08-05 Sist oppdatert: 2017-12-11bibliografisk kontrollert
    2. Chromo domain proteins in balanced dosage together with boundary elements cooperate in organising the mating-type chromatin in fission yeast
    Åpne denne publikasjonen i ny fane eller vindu >>Chromo domain proteins in balanced dosage together with boundary elements cooperate in organising the mating-type chromatin in fission yeast
    (engelsk)Manuskript (Annet (populærvitenskap, debatt, mm))
    Abstract [en]

    The chromatin in the cell nucleus has a spatial organisation. For example, in the fission yeast, Schizosaccharomyces pombe, transcriptionally repressed heterochromatin is found at the nuclear membrane (NM). The centromeres and the mating-type region localise in the proximity of the spindle pole body (SPB), while the telomeres are found on the opposite side of the nucleus in the proximity of the nucleolus. In a previous study we used the mating-type region as a model to study the driving force behind nuclear organisation. We proposed two mutually exclusive models to explain what determines the localisation of the mating-type region. The first model suggests that solely the amount of heterochromatin in the region affects the localisation, while the other model stipulates that the boundary elements together with heterochromatin formation anchor the mating-type region in the NM in the vicinity of the SPB. Here, we present data that disproves the first model. We found that in a strain expressing tripled amounts of the chromodomain protein Swi6, a structural component of heterochromatin, the mating-type region was delocalised from the proximity of the SPB. A strain deleted of the histone deacetylase clr3+ also had a delocalised mating-type locus. Interestingly, a strain with a point-mutation in clr3-735 producing an enzymatically inactive protein in normal amounts showed an intermediate phenotype. Most importantly, we identify the chromodomain proteins, Chp1 and Chp2, as crucial factors for correct subnuclear localisation of the mating-type region. We suggest that boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin.

    Identifikatorer
    urn:nbn:se:uu:diva-107280 (URN)
    Tilgjengelig fra: 2009-08-04 Laget: 2009-08-04 Sist oppdatert: 2010-01-14bibliografisk kontrollert
    3. Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe
    Åpne denne publikasjonen i ny fane eller vindu >>Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe
    Vise andre…
    2009 (engelsk)Inngår i: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 118, nr 1, s. 99-112Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    There are several documented events of changes in subnuclear localization during gene activation. However, there are conflicting data on whether the nuclear periphery is a compartment for gene repression or activation, and whether genes are moved to the pores at the nuclear membrane (NM) or not during gene activation. Nitrogen starvation of fission yeast serves as a good model system for studying gene induction since it causes fast regulation of hundreds of genes. In this study the subnuclear localization of two gene clusters repressed by nitrogen was investigated. During normal growth conditions the gene clusters localized to the nuclear periphery at the opposite side of the nucleus as compared to the spindle pole body (SPB). This constrained localization was dependent on the histone deacetylase Clr3, known to transcriptionally repress genes in these clusters. Already 20 minutes after nitrogen depletion drastic changes in subnuclear localization of the two loci were observed, away from the NM towards the nuclear interior. At least for one of the clusters the movement was clearly transcription dependent. Data presented here illustrates how interconnected events of gene activation and nuclear reorganization are, as well as provides a suggestion of how nuclear organization might be maintained.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-107289 (URN)10.1007/s00412-008-0180-6 (DOI)000262504300009 ()
    Tilgjengelig fra: 2009-08-05 Laget: 2009-08-05 Sist oppdatert: 2017-12-13bibliografisk kontrollert
  • 24. Alizadehheidari, Mohammadreza
    et al.
    Werner, Erik
    Noble, Charleston
    Reiter-Schad, Michaela
    Nyberg, Lena K.
    Fritzsche, Joachim
    Mehlig, Bernhard
    Tegenfeldt, Jonas O.
    Ambjornsson, Tobias
    Persson, Fredrik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Westerlund, Fredrik
    Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics2015Inngår i: Macromolecules, ISSN 0024-9297, E-ISSN 1520-5835, Vol. 48, nr 3, s. 871-878Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of confined DNA from the circular to linear configuration as a light-induced double-strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to what extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations.

  • 25. Allahverdiyeva, Yagut
    et al.
    Mamedov, Fikret
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström.
    Mäenpää, Pirkko
    Vass, Imre
    Aro, Eva-Mari
    Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: characterization of the rbcL deletion mutant of tobacco2005Inngår i: Biochimica et Biophysica Acta (BBA) - Bioenergetics, ISSN 0005-2728, Vol. 1709, nr 1, s. 69-83Artikkel i tidsskrift (Fagfellevurdert)
  • 26.
    Almandoz Gil, Leire
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap.
    Characterization of Physiological and Pathological Alpha-Synuclein: Implications for Parkinson’s Disease and Related Disorders2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Aggregated alpha-synuclein is the main component of Lewy bodies and Lewy neurites, intraneuronal inclusions found in the brains of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) patients (synucleinopathies). Alpha-synuclein is a presynaptic protein, which is most commonly an unfolded monomer in its physiological state. However, under pathological conditions it can start to misfold and enter an aggregation pathway that will lead to the formation of oligomers of increasing size and finally insoluble fibrils. The oligomers have been hypothesized to be the most neurotoxic species, but studies of their properties have been hindered by their heterogeneity and kinetic instability. The overall aim of this thesis was to characterize and compare physiological and pathological forms of alpha-synuclein from different sources: recombinant monomers, oligomers formed in vitro through exposure to oxidative stress related reactive aldehydes, aggregates from a synucleinopathy mouse model and from synucleinopathy patients.

    In paper I we studied the effect of low molar excess of two lipid peroxidation products, 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE), on the oligomerization of alpha-synuclein. Through biophysical methods we observed that, although both aldehydes bound to alpha-synuclein directly, ONE produced SDS-stable oligomers more rapidly than HNE. Moreover, ONE induced oligomerization at both acidic and neutral pH, while HNE only formed oligomers at neutral pH.

    In paper II we mapped the surface exposed epitopes of in vitro and in vivo generated alpha-synuclein species by using immunoglobulin Y antibodies raised against short linear peptides covering most of the alpha-synuclein sequence. Monomers were found to react with most antibodies, while the latter part of the N-terminus and mid-region of HNE oligomers and fibrils was found to be occluded in oligomers and fibrils. Through immunohistochemistry we compared alpha-synuclein aggregates in brain tissue from patients with synucleinopathies as well as from a mouse model expressing A30P human alpha-synuclein. Although the exposed epitopes were found to be similar overall, subtle differences were detected in the C-terminus.

    An additional aim of this thesis was to characterize synaptic aggregates of alpha-synuclein. In paper III we obtained synaptosomal preparations of the A30P mouse model and found that a subset of the alpha-synuclein present in the synaptosomes was proteinase K resistant and therefore aggregated. Further biochemical analyses showed that the aggregated alpha-synuclein mainly was of human, i.e. transgenic, origin and that Ser 129 was not phosphorylated, which otherwise is a common post translational modification of alpha-synuclein in Lewy bodies.

    It has been suggested that alpha-synuclein plays a role in neurotransmitter release by binding to the SNARE protein VAMP-2 and thereby chaperoning the SNARE complex assembly. In paper IV we used proximity ligation assay to visualize the co-localization of alpha-synuclein and the SNARE proteins in primary neurons from non-transgenic and A30P transgenic mice.

    In conclusion, in this thesis we have characterized a variety of alpha-synuclein species and shed light on the diversity of alpha-synuclein aggregates. Additionally, we have characterized synaptic species of alpha-synuclein and analyzed the co-localization between alpha-synuclein and SNARE proteins in neurons.

    Delarbeid
    1. Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways
    Åpne denne publikasjonen i ny fane eller vindu >>Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways
    Vise andre…
    2017 (engelsk)Inngår i: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 110, s. 421-431Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.

    Emneord
    Alpha-synuclein, Oligomers, 4-oxo-2-nonenal, 4-hydroxy-2-nonenal, Oxidative stress
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-326663 (URN)10.1016/j.freeradbiomed.2017.07.004 (DOI)000406049200038 ()28690195 (PubMedID)
    Forskningsfinansiär
    Swedish Research Council, 2011-4519, 2012-2172, 2010-6745Marianne and Marcus Wallenberg FoundationThe Swedish Brain FoundationSwedish Society of MedicineÅke Wiberg Foundation
    Merknad

    Correction in: Free Radical Biology and Medicine, vol. 117, pages 258-258.

    DOI: 10.1016/j.freeradbiomed.2018.02.007

    Tilgjengelig fra: 2017-07-19 Laget: 2017-07-19 Sist oppdatert: 2018-07-27bibliografisk kontrollert
    2. Mapping of Surface-Exposed Epitopes of In Vitro and In Vivo Aggregated Species of Alpha-Synuclein
    Åpne denne publikasjonen i ny fane eller vindu >>Mapping of Surface-Exposed Epitopes of In Vitro and In Vivo Aggregated Species of Alpha-Synuclein
    Vise andre…
    2017 (engelsk)Inngår i: Cellular and molecular neurobiology, ISSN 0272-4340, E-ISSN 1573-6830, Vol. 37, nr 7, s. 1217-1226Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal deposits observed in Parkinson's disease and dementia with Lewy bodies. The objective of the study was to identify surface-exposed epitopes of alpha-synuclein in vitro and in vivo formed aggregates. Polyclonal immunoglobulin Y antibodies were raised against short linear peptides of the alpha-synuclein molecule. An epitope in the N-terminal region (1-10) and all C-terminal epitopes (90-140) were found to be exposed in an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant monomeric, oligomeric, and fibrillar alpha-synuclein. In a phospholipid ELISA, the N-terminus and mid-region of alpha-synuclein (i.e., 1-90) were associated with phosphatidylserine and thus occluded from antibody binding. The antibodies that reacted most strongly with epitopes in the in vitro aggregates (i.e., 1-10 and epitopes between positions 90-140) also labeled alpha-synuclein inclusions in brains from transgenic (Thy-1)-h[A30P] alpha-synuclein mice and Lewy bodies and Lewy neurites in brains of patients with alpha-synucleinopathies. However, differences in reactivity were observed with the C-terminal antibodies when brain tissue from human and transgenic mice was compared. Taken together, the study shows that although similar epitopes are exposed in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity can be observed between different molecular species.

    Emneord
    Parkinson's disease, Dementia with Lewy bodies, Alpha-synuclein, Epitope mapping
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-334384 (URN)10.1007/s10571-016-0454-0 (DOI)000409352200007 ()
    Tilgjengelig fra: 2017-12-15 Laget: 2017-12-15 Sist oppdatert: 2018-02-23bibliografisk kontrollert
    3. Characterization of synaptic aggregates of alpha-synuclein in (Thy-1)-h[A30P] alpha-synuclein mice
    Åpne denne publikasjonen i ny fane eller vindu >>Characterization of synaptic aggregates of alpha-synuclein in (Thy-1)-h[A30P] alpha-synuclein mice
    Vise andre…
    (engelsk)Manuskript (preprint) (Annet vitenskapelig)
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-342341 (URN)
    Tilgjengelig fra: 2018-02-20 Laget: 2018-02-20 Sist oppdatert: 2018-02-23
    4. In situ proximity ligation assay reveals co-localization of alpha-synuclein and SNARE proteins in murine primary neurons
    Åpne denne publikasjonen i ny fane eller vindu >>In situ proximity ligation assay reveals co-localization of alpha-synuclein and SNARE proteins in murine primary neurons
    Vise andre…
    2018 (engelsk)Inngår i: Frontiers in Neurology, ISSN 1664-2295, E-ISSN 1664-2295, Vol. 9, artikkel-id 180Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The aggregation of alpha-synuclein (alpha Syn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of aSyn is to promote the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between aSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human aSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human aSyn, a PLA signal indicating close proximity between aSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human aSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of aSyn and the SNARE proteins in primary neuronal cultures

    sted, utgiver, år, opplag, sider
    Frontiers Media S.A., 2018
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-342580 (URN)10.3389/fneur.2018.00180 (DOI)000428063500001 ()29623065 (PubMedID)
    Forskningsfinansiär
    Marianne and Marcus Wallenberg FoundationThe Swedish Brain FoundationSwedish Society of MedicineMagnus Bergvall Foundation
    Tilgjengelig fra: 2018-02-22 Laget: 2018-02-22 Sist oppdatert: 2018-06-28bibliografisk kontrollert
  • 27.
    Almandoz-Gil, Leire
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Welander, Hedvig
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Ihse, Elisabet
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Khoonsari, Payam Emami
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin.
    Musunuri, Sravani
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi.
    Lendel, Christofer
    KTH Royal Inst Technol, Dept Chem, Stockholm, Sweden.
    Sigvardson, Jessica
    BioArctic AB, Stockholm, Sweden.
    Karlsson, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Tillämpad materialvetenskap.
    Ingelsson, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Kultima, Kim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin.
    Bergström, Joakim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Corrigendum to “Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways” [Free Rad. Biol. Med. (2017) 421–431]2018Inngår i: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 117, s. 258-258Artikkel i tidsskrift (Fagfellevurdert)
  • 28.
    Almandoz-Gil, Leire
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Welander, Hedvig
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Ihse, Elisabet
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Khoonsari, Payam Emami
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin.
    Musunuri, Sravani
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi.
    Lendel, Christofer
    KTH, Royal Institute of Technology, Sweden.
    Sigvardson, Jessica
    BioArctic AB, Sweden.
    Karlsson, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Tekniska sektionen, Institutionen för teknikvetenskaper, Tillämpad materialvetenskap.
    Ingelsson, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Kultima, Kim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Cancerfarmakologi och beräkningsmedicin.
    Bergström, Joakim
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
    Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways2017Inngår i: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 110, s. 421-431Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.

  • 29.
    Almaqwashi, Ali A.
    et al.
    Northeastern Univ, Dept Phys, Boston, MA 02115 USA.;King Abdulaziz Univ, Dept Phys, Rabigh 21911, Saudi Arabia..
    Andersson, Johanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC. Chalmers, Dept Chem & Chem Engn, SE-41296 Gothenburg, Sweden..
    Lincoln, Per
    Chalmers, Dept Chem & Chem Engn, SE-41296 Gothenburg, Sweden..
    Rouzina, Ioulia
    Ohio State Univ, Dept Chem & Biochem, Columbus, OH 43210 USA..
    Westerlund, Fredrik
    Chalmers, Dept Biol & Biol Engn, SE-41296 Gothenburg, Sweden..
    Williams, Mark C.
    Northeastern Univ, Dept Phys, Boston, MA 02115 USA..
    DNA intercalation optimized by two-step molecular lock mechanism2016Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikkel-id 37993Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The diverse properties of DNA intercalators, varying in affinity and kinetics over several orders of magnitude, provide a wide range of applications for DNA-ligand assemblies. Unconventional intercalation mechanisms may exhibit high affinity and slow kinetics, properties desired for potential therapeutics. We used single-molecule force spectroscopy to probe the free energy landscape for an unconventional intercalator that binds DNA through a novel two-step mechanism in which the intermediate and final states bind DNA through the same mono-intercalating moiety. During this process, DNA undergoes significant structural rearrangements, first lengthening before relaxing to a shorter DNA-ligand complex in the intermediate state to form a molecular lock. To reach the final bound state, the molecular length must increase again as the ligand threads between disrupted DNA base pairs. This unusual binding mechanism results in an unprecedented optimized combination of high DNA binding affinity and slow kinetics, suggesting a new paradigm for rational design of DNA intercalators.

  • 30.
    Almén, Markus Sällman
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Lamichhaney, Sangeet
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Berglund, Jonas
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Grant, B. Rosemary
    Princeton Univ, Dept Ecol & Evolutionary Biol, Princeton, NJ 08544 USA..
    Grant, Peter R.
    Princeton Univ, Dept Ecol & Evolutionary Biol, Princeton, NJ 08544 USA..
    Webster, Matthew T.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Andersson, Leif
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Swedish Univ Agr Sci, Dept Anim Breeding & Genet, Uppsala, Sweden.;Texas A&M Univ, Dept Vet Integrat Biosci, College Stn, TX USA..
    Adaptive radiation of Darwin's finches revisited using whole genome sequencing2016Inngår i: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 38, nr 1, s. 14-20Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We recently used genome sequencing to study the evolutionary history of the Darwin's finches. A prominent feature of our data was that different polymorphic sites in the genome tended to indicate different genetic relationships among these closely related species. Such patterns are expected in recently diverged genomes as a result of incomplete lineage sorting. However, we uncovered conclusive evidence that these patterns have also been influenced by interspecies hybridisation, a process that has likely played an important role in the radiation of Darwin's finches. A major discovery was that segregation of two haplotypes at the ALX1 locus underlies variation in beak shape among the Darwin's finches, and that differences between the two haplotypes in a 240 kb region in blunt and pointed beaked birds involve both coding and regulatory changes. As we review herein, the evolution of such adaptive haplotypes comprising multiple causal changes appears to be an important mechanism contributing to the evolution of biodiversity.

  • 31.
    Altai, Mohamed
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk strålningsvetenskap.
    Liu, Hao
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Ding, Haozhong
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Mitran, Bogdan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Theranostics.
    Edqvist, Per-Henrik D
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Experimentell och klinisk onkologi.
    Tolmachev, Vladimir
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk strålningsvetenskap.
    Orlova, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Medicinsk strålningsvetenskap.
    Gräslund, Torbjorn
    KTH Royal Inst Technol, Dept Prot Sci, Roslagstullsbacken 21, S-11417 Stockholm, Sweden.
    Affibody-derived drug conjugates: Potent cytotoxic molecules for treatment of HER2 over-expressing tumors2018Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 288, s. 84-95Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Patients with HER2-positive tumors often suffer resistance to therapy, warranting development of novel treatment modalities. Affibody molecules are small affinity proteins which can be engineered to bind to desired targets. They have in recent years been found to allow precise targeting of cancer specific molecular signatures such as the HER2 receptor. In this study, we have investigated the potential of an affibody molecule targeting HER2, Z(HER2:2891), conjugated with the cytotoxic maytansine derivate MC-DM1, for targeted cancer therapy. Z(HER2:2891) was expressed as a monomer (Z(HER2:2891)), dimer ((Z(HER2:2891)) 2) and dimer with an albumin binding domain (ABD) for half-life extension ((Z(HER2:2891)) 2-ABD). All proteins had a unique C-terminal cysteine that could be used for efficient and site-specific conjugation with MC-DM1. The resulting affibody drug conjugates were potent cytotoxic molecules for human cells over-expressing HER2, with sub-nanomolar IC50-values similar to trastuzumab emtansine, and did not affect cells with low HER2 expression. A biodistribution study of a radiolabeled version of (Z(HER2:2891))(2)-ABD-MC-DM1, showed that it was taken up by the tumor. The major site of off-target uptake was the kidneys and to some extent the liver. (Z(HER2:2891)) 2-ABD-MC-DM1 was found to have a half-life in circulation of 14 h. The compound was tolerated well by mice at 8.5 mg/kg and was shown to extend survival of mice bearing HER2 over-expressing tumors. The findings in this study show that affibody molecules are a promising class of engineered affinity proteins to specifically deliver small molecular drugs to cancer cells and that such conjugates are potential candidates for clinical evaluation on HER2-overexpressing cancers.

  • 32.
    Altuvia, S.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Wagner, E.G.H.
    Switching on and off with RNA.2000Inngår i: Proc. Natl. Acad. Sci. USA, Vol. 97, nr 18, s. 9824-9826Artikkel i tidsskrift (Fagfellevurdert)
  • 33.
    Al-walai, Somar
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för biologisk grundutbildning. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för neurovetenskap.
    Characterization of solutecarrier SLC38A62012Student paper other, 10 poäng / 15 hpOppgave
    Abstract [en]

    Transport across the membrane of a cell is of crucial importance for cellular functions. The solute carrier family,SLC38 is a family of membrane proteins that transports various substances through the membrane and thusperforms many physiologically important functions, for example, transport of glutamine from astrocyte toneurons in the central nervous system. In this paper, we demonstrate that one of the transporters in this familynamed SLC38A6 forms several protein complexes with a variety of proteins in the membrane and in synapticvesicles, suggesting that SLC38A6 is involved in the synaptic release of neurotransmitters in synapses. Weperformed sensitive protein interaction analysis between the protein of interest and a variety of proteinsexpressed at different sites in the neuronal cell. We showed that SLC38A6 interacts with proteins in the cellmembrane as well as in the membrane of synaptic vesicles. The current theory is that SLC38A6 interact withthese proteins when the synaptic vesicles are in close proximity with the cell membrane during the release of theneurotransmitters.

  • 34.
    Ament-Velasquez, Sandra Lorena
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Systematisk biologi. Univ Montpellier, Inst Evolutionary Sci, CNRS, IRD,EPHE, Pl Eugene Bataillon, F-34095 Montpellier, France..
    Figuet, E.
    Univ Montpellier, Inst Evolutionary Sci, CNRS, IRD,EPHE, Pl Eugene Bataillon, F-34095 Montpellier, France..
    Ballenghien, M.
    Univ Montpellier, Inst Evolutionary Sci, CNRS, IRD,EPHE, Pl Eugene Bataillon, F-34095 Montpellier, France..
    Zattara, E. E.
    Indiana Univ, Dept Biol, 107 S Indiana Ave, Bloomington, IN 47405 USA.;Smithsonian Inst, Natl Museum Nat Hist, Dept Invertebrate Zool, 10th St & Constitut Ave NW, Washington, DC 20560 USA..
    Norenburg, J. L.
    Smithsonian Inst, Natl Museum Nat Hist, Dept Invertebrate Zool, 10th St & Constitut Ave NW, Washington, DC 20560 USA..
    Fernandez-Alvarez, F. A.
    CSIC Barcelona, Inst Ciencies Mar, Barcelona 08003, Spain..
    Bierne, J.
    Univ Reims, Lab Biol Cellulaire & Mol, 9 Blvd Paix, F-51100 Reims, France..
    Bierne, N.
    Univ Montpellier, Inst Evolutionary Sci, CNRS, IRD,EPHE, Pl Eugene Bataillon, F-34095 Montpellier, France..
    Galtier, N.
    Univ Montpellier, Inst Evolutionary Sci, CNRS, IRD,EPHE, Pl Eugene Bataillon, F-34095 Montpellier, France..
    Population genomics of sexual and asexual lineages in fissiparous ribbon worms (Lineus, Nemertea): hybridization, polyploidy and the Meselson effect2016Inngår i: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 25, nr 14, s. 3356-3369Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Comparative population genetics in asexual vs. sexual species offers the opportunity to investigate the impact of asexuality on genome evolution. Here, we analyse coding sequence polymorphism and divergence patterns in the fascinating Lineus ribbon worms, a group of marine, carnivorous nemerteans with unusual regeneration abilities, and in which asexual reproduction by fissiparity is documented. The population genomics of the fissiparous L. pseudolacteus is characterized by an extremely high level of heterozygosity and unexpectedly elevated pi(N)/pi(S) ratio, in apparent agreement with theoretical expectations under clonal evolution. Analysis of among-species allele sharing and read-count distribution, however, reveals that L. pseudolacteus is a triploid hybrid between Atlantic populations of L. sanguineus and L. lacteus. We model and quantify the relative impact of hybridity, polyploidy and asexuality on molecular variation patterns in L. pseudolacteus and conclude that (i) the peculiarities of L. pseudolacteus population genomics result in the first place from hybridization and (ii) the accumulation of new mutations through the Meselson effect is more than compensated by processes of heterozygosity erosion, such as gene conversion or gene copy loss. This study illustrates the complexity of the evolutionary processes associated with asexuality and identifies L. pseudolacteus as a promising model to study the first steps of polyploid genome evolution in an asexual context.

  • 35.
    Amiri, H.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolutionsbiologi, Molekylär evolution.
    Alsmark, U. C. M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolutionsbiologi, Molekylär evolution.
    Andersson, S. G. E.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolutionsbiologi, Molekylär evolution.
    Proliferation and deterioration of Rickettsia palindromic elements2002Inngår i: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 19, nr 8, s. 1234-1243Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    It has been suggested that Rickettsia Palindromic Elements (RPEs) have evolved as selfish DNA that mediate protein sequence evolution by being targeted to genes that code for RNA and proteins. Here, we have examined the phylogenetic depth of two RPEs that are located close to the genes encoding elongation factors Tu (tuf) and G (fus) in Rickettsia. An exceptional organization of the elongation factor genes was found in all 11 species examined, with complete or partial RPEs identified downstream of the tuf gene (RPE-tuf) in six species and of the fus gene (RPE-fus) in 10 species. A phylogenetic reconstruction shows that both RPE-tuf and RPE-fus have evolved in a manner that is consistent with the expected species divergence. The analysis provides evidence for independent loss of RPE-tuf in several species, possibly mediated by short repetitive sequences flanking the site of excision. The remaining RPE-tuf sequences evolve as neutral sequences in different stages of deterioration. Likewise, highly fragmented remnants of the RPE-fus sequence were identified in two species. This suggests that genome-specific differences in the content of RPEs are the result of recent loss rather than recent proliferation.

  • 36.
    Amiri, H.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för evolutionsbiologi. MOLECULAR EVOLUTION.
    Andersson, S. G. E.
    Rickettsia: The highly rearranged cousin of mitochondria.2001Inngår i: Recent Res. Devel. Microbiology, Vol. 5, s. 321-329Artikkel i tidsskrift (Fagfellevurdert)
  • 37.
    Amiri, H.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för evolutionsbiologi. MOLECULAR EVOLUTION.
    Davids, W.
    Andersson, S.G.E.
    Birth and death of orphan genes in Rickettsia.2003Inngår i: Mol. Biol. Evol., Vol. 20, s. 1575-1587Artikkel i tidsskrift (Fagfellevurdert)
  • 38.
    Amiri, H.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för evolutionsbiologi. MOLECULAR EVOLUTION.
    Karlberg, O.
    Andersson, S.G.E.
    Deep origin of plastid/parasite ATP/ADP translocases.2003Inngår i: J Mol Evol, Vol. 56, s. 137-150Artikkel i tidsskrift (Fagfellevurdert)
  • 39.
    Amrein, Beat A.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Naworyta, Agata
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Biokemi.
    Kamerlin, Shina C. L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015Inngår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, nr 10, s. 5702-5713Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

  • 40.
    Amrein, Beat Anton
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Extending the Reach of Computational Approaches to Model Enzyme Catalysis2017Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically:

    1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems.

    2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1.

    3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example.

    In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

    Delarbeid
    1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Åpne denne publikasjonen i ny fane eller vindu >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Vise andre…
    2014 (engelsk)Inngår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, nr 16, s. 4351-4362Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Forskningsfinansiär
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Tilgjengelig fra: 2014-06-23 Laget: 2014-06-04 Sist oppdatert: 2018-12-03bibliografisk kontrollert
    2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Åpne denne publikasjonen i ny fane eller vindu >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Vise andre…
    2015 (engelsk)Inngår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, nr 10, s. 5702-5713Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

    HSV kategori
    Forskningsprogram
    Biokemi
    Identifikatorer
    urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
    Forskningsfinansiär
    EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Tilgjengelig fra: 2015-08-18 Laget: 2015-08-18 Sist oppdatert: 2017-12-04bibliografisk kontrollert
    3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Åpne denne publikasjonen i ny fane eller vindu >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Vise andre…
    2016 (engelsk)Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, nr 24, s. 5639-5651Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
    Forskningsfinansiär
    Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
    Tilgjengelig fra: 2016-04-01 Laget: 2016-04-01 Sist oppdatert: 2017-11-30bibliografisk kontrollert
    4. CADEE: Computer-Aided Directed Evolution of Enzymes
    Åpne denne publikasjonen i ny fane eller vindu >>CADEE: Computer-Aided Directed Evolution of Enzymes
    Vise andre…
    2017 (engelsk)Inngår i: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, nr 1, s. 50-64Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

    Emneord
    computational directed evolution, computational enzyme design, distributed computing, empirical valence bond, triosephosphate isomerase
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-314218 (URN)10.1107/S2052252516018017 (DOI)000392925800007 ()
    Forskningsfinansiär
    EU, FP7, Seventh Framework Programme, 306474Knut and Alice Wallenberg FoundationThe Royal Swedish Academy of SciencesSwedish Research Council, 2015-04928Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Tilgjengelig fra: 2017-01-31 Laget: 2017-01-31 Sist oppdatert: 2018-01-13bibliografisk kontrollert
  • 41.
    Andaloussi, Mounir
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Henriksson, Lena M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Wieckowska, Anna
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Lindh, Martin
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Björkelid, Christofer
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Larsson, Anna M.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Suresh, Surisetti
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Iyer, Harini
    Srinivasa, Bachally R.
    Bergfors, Terese
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Unge, Torsten
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Mowbray, Sherry L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Larhed, Mats
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Jones, T. Alwyn
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Karlén, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase2011Inngår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, nr 14, s. 4964-4976Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

  • 42. Andersen, Felicie F.
    et al.
    Knudsen, Bjarne
    Oliveira, Cristiano Luis Pinto
    Frøhlich, Rikke F.
    Krüger, Dinna
    Bungert, Jörg
    Agbandje-McKenna, Mavis
    McKenna, Robert
    Juul, Sissel
    Veigaard, Christopher
    Koch, Jørn
    Rubinstein, John L.
    Guldbrandtsen, Bernt
    Hede, Marianne S.
    Karlsson, Göran
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för fysikalisk och analytisk kemi, Fysikalisk kemi.
    Andersen, Anni H.
    Pedersen, Jan Skov
    Knudsen, Birgitta R.
    Assembly and structural analysis of a covalently closed nano-scale DNA cage2008Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, nr 4, s. 1113-1119Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The inherent properties of DNA as a stable polymer with unique affinity for partner mols. detd. by the specific Watson-Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnol. Here we present the design, construction and structural anal. of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helixes that assembles from eight oligonucleotides with a yield of .apprx.30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-mols. to enable their investigation in certain harmful environments or even allow their organization into higher order structures.

  • 43. Andersen, Gorm
    et al.
    Andersen, Birgit
    Dobritzsch, Doreen
    Karolinska Institutet.
    Schnackerz, Klaus D
    Piskur, Jure
    A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast2007Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, nr 7, s. 1804-1017Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.

  • 44.
    Andersson, Anders
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Bernander, Rolf
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Nilsson, Peter
    Dual-genome primer design for construction of DNA microarrays2005Inngår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 21, nr 3, s. 325-332Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Motivation: Microarray experiments using probes covering a whole transcriptome are expensive to initiate, and a major part of the costs derives from synthesizing gene-specific PCR primers or hybridization probes. The high costs may force researchers to limit their studies to a single organism, although comparing gene expression in different species would yield valuable information.

    Results: We have developed a method, implemented in the software DualPrime, that reduces the number of primers required to amplify the genes of two different genomes. The software identifies regions of high sequence similarity, and from these regions selects PCR primers shared between the genomes, such that either one or, preferentially, both primers in a given PCR can be used for amplification from both genomes. To assure high microarray probe specificity, the software selects primer pairs that generate products of low sequence similarity to other genes within the same genome. We used the software to design PCR primers for 2182 and 1960 genes from the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. Primer pairs were shared among 705 pairs of genes, and single primers were shared among 1184 pairs of genes, resulting in a saving of 31% compared to using only unique primers. We also present an alternative primer design method, in which each gene shares primers with two different genes of the other genome, enabling further savings.

  • 45.
    Andersson, C. Evalena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Structure-Function Studies of Enzymes from Ribose Metabolism2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    In the pentose phosphate pathway, carbohydrates such as glucose and ribose are degraded with production of reductive power and energy. Another important function is to produce essential pentoses, such as ribose 5-phosphate, which later can be used in biosynthesis of nucleic acids and cofactors.

    This thesis presents structural and functional studies on three enzymes involved in ribose metabolism in Escherichia coli.

    Ribokinase is an enzyme that phosphorylates ribose in the presence of ATP and magnesium, as the first step of exogenous ribose metabolism. Two important aspects of ribokinase function, not previously known, have been elucidated. Ribokinase was shown to be activated by monovalent cations, specifically potassium. Structural analysis of the monovalent ion binding site indicates that the ion has a structural rather than catalytic role; a mode of activation involving a conformational change has been suggested. Product inhibition studies suggest that ATP is the first substrate to bind the enzyme. Independent Kd measurements with the ATP analogue AMP-PCP support this. The results presented here will have implications for several enzymes in the protein family to which ribokinase belongs, in particular the medically interesting enzyme adenosine kinase.

    Ribose 5-phosphate isomerases convert ribose 5-phosphate into ribulose 5-phosphate or vice versa. Structural studies on the two genetically distinct isomerases in E. coli have shown them to be fundamentally different in many aspects, including active site architecture. However, a kinetic study has demonstrated both enzymes to be efficient in terms of catalysis. Sequence searches of completed genomes show ribose 5-phosphate isomerase B to be the sole isomerase in many bacteria, although ribose 5-phosphate isomerase A is a nearly universal enzyme. All genomes contain at least one of the two enzymes. These results confirm that both enzymes must be independently capable of supporting ribose metabolism, a fact that had not previously been established.

    Delarbeid
    1. Activation of Ribokinase by Monovalent Cations
    Åpne denne publikasjonen i ny fane eller vindu >>Activation of Ribokinase by Monovalent Cations
    2002 (engelsk)Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 315, nr 3, s. 409-419Artikkel i tidsskrift (Fagfellevurdert) Published
    HSV kategori
    Identifikatorer
    urn:nbn:se:uu:diva-91364 (URN)10.1006/jmbi.2001.5248 (DOI)
    Tilgjengelig fra: 2004-02-12 Laget: 2004-02-12 Sist oppdatert: 2017-12-14bibliografisk kontrollert
    2. Structure of Escherichia coli Ribose 5-phosphate Isomerase: A Ubiquitous Enzyme of the Pentose Phosphate Pathway and the Calvin Cycle
    Åpne denne publikasjonen i ny fane eller vindu >>Structure of Escherichia coli Ribose 5-phosphate Isomerase: A Ubiquitous Enzyme of the Pentose Phosphate Pathway and the Calvin Cycle
    Vise andre…
    2003 Inngår i: Structure, Vol. 11, nr 1, s. 31-42Artikkel i tidsskrift (Fagfellevurdert) Published
    Identifikatorer
    urn:nbn:se:uu:diva-91365 (URN)
    Tilgjengelig fra: 2004-02-12 Laget: 2004-02-12bibliografisk kontrollert
    3. The 2.2 Å Resolution Structure of RpiB/AlsB from Escherichia coli Illutrates a New Approach to the Ribose 5-phosphate Isomerase Reaction
    Åpne denne publikasjonen i ny fane eller vindu >>The 2.2 Å Resolution Structure of RpiB/AlsB from Escherichia coli Illutrates a New Approach to the Ribose 5-phosphate Isomerase Reaction
    Vise andre…
    2003 Inngår i: Journal of Molecular Biology, Vol. 332, nr 5, s. 1083-1094Artikkel i tidsskrift (Fagfellevurdert) Published
    Identifikatorer
    urn:nbn:se:uu:diva-91366 (URN)
    Tilgjengelig fra: 2004-02-12 Laget: 2004-02-12bibliografisk kontrollert
    4. Specificity and Activity of Escherichia coli Ribokinase
    Åpne denne publikasjonen i ny fane eller vindu >>Specificity and Activity of Escherichia coli Ribokinase
    Manuskript (Annet vitenskapelig)
    Identifikatorer
    urn:nbn:se:uu:diva-91367 (URN)
    Tilgjengelig fra: 2004-02-12 Laget: 2004-02-12 Sist oppdatert: 2016-05-09bibliografisk kontrollert
    5. Mycobacterium tuberculosis Ribose 5-phosphate Isomerase has a Known Fold, but a Novel Active Site
    Åpne denne publikasjonen i ny fane eller vindu >>Mycobacterium tuberculosis Ribose 5-phosphate Isomerase has a Known Fold, but a Novel Active Site
    Vise andre…
    2004 Inngår i: Journal of Molecular Biology, Vol. 335, nr 3, s. 799-809Artikkel i tidsskrift (Fagfellevurdert) Published
    Identifikatorer
    urn:nbn:se:uu:diva-91368 (URN)
    Tilgjengelig fra: 2004-02-12 Laget: 2004-02-12bibliografisk kontrollert
  • 46.
    Andersson, Hans O.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Fridborg, Kerstin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Löwgren, Seved
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Alterman, Mathias
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Mühlman, Anna
    Björsne, Magnus
    Garg, Neeraj
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Kvarnström, Ingemar
    Schaal, Wesley
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Classon, Björn
    Karlén, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Danielson, U. Helena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
    Ahlsén, Göran
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
    Nillroth, Ulrika
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi.
    Vrang, Lotta
    Öberg, Bo
    Samuelsson, Bertil
    Hallberg, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för organisk farmaceutisk kemi.
    Unge, Torsten
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors2003Inngår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, nr 8, s. 1746-1758Artikkel i tidsskrift (Fagfellevurdert)
  • 47.
    Andersson, Inger
    et al.
    Department of Molecular Biology, Swedish University of Agricultural Sciences.
    Backlund, Anders
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
    Structure and function of Rubisco2008Inngår i: Plant physiology and biochemistry (Paris), ISSN 0981-9428, E-ISSN 1873-2690, Vol. 46, nr 3, s. 275-291Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO2 into the biosphere. At the same time Rubisco is an extremely inefficient catalyst and its carboxylase activity is compromised by an opposing oxygenase activity involving atmospheric O2. The shortcomings of Rubisco have implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. This review uses the information provided in these structures in a structure-based sequence alignment and discusses Rubisco function in the context of structural variations at all levels – amino acid sequence, fold, tertiary and quaternary structure – with an evolutionary perspective and an emphasis on the structural features of the enzyme that may determine its function as a carboxylase.

  • 48.
    Andersson, Jan O.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolution, genomik och systematik, Molekylär evolution.
    Eukaryotic gene transfer: adaptation and replacements2008Inngår i: Horizontal Gene Transfer in the Evolution of Pathogenesis / [ed] Michael Hensel, Herbert Schmidt, Cambridge: Cambridge University Press , 2008, s. 293-316Kapittel i bok, del av antologi (Annet vitenskapelig)
  • 49.
    Andersson, Jan O
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolutionsbiologi.
    Andersson, Siv GE
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för evolutionsbiologi.
    Pseudogenes, junk DNA, and the dynamics of Rickettsia genomes2001Inngår i: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 18, nr 5, s. 829-839Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Studies of neutrally evolving sequences suggest that differences in eukaryotic genome sizes result from different rates of DNA loss. However, very few pseudogenes have been identified in microbial species, and the processes whereby genes and genomes deteriorate in bacteria remain largely unresolved. The typhus-causing agent, Rickettsia prowazekii, is exceptional in that as much as 24% of its 1.1-Mb genome consists of noncoding DNA and pseudogenes. To test the hypothesis that the noncoding DNA in the R. prowazekii genome represents degraded remnants of ancestral genes, we systematically examined all of the identified pseudogenes and their flanking sequences in three additional Rickettsia species. Consistent with the hypothesis, we observe sequence similarities between genes and pseudogenes in one species and intergenic DNA in another species. We show that the frequencies and average sizes of deletions are larger than insertions in neutrally evolving pseudogene sequences. Our results suggest that inactivated genetic material in the Rickettsia genomes deteriorates spontaneously due to a mutation bias for deletions and that the noncoding sequences represent DNA in the final stages of this degenerative process.

  • 50.
    Andersson, Malena
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi. Institutionen för biokemi och organisk kemi, Biokemi.
    Mannervik, Bengt
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för naturvetenskaplig biokemi. Institutionen för biokemi och organisk kemi, Biokemi.
    Exploring protein evolution by saturation mutagenesis of the GST M2-2 active site residue 2102005Inngår i: FEBS Journal, Vol. 272, s. 81 Suppl-Artikkel i tidsskrift (Annet vitenskapelig)
1234567 1 - 50 of 1624
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