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  • 101. Haldimann, D
    et al.
    Brodelius, Peter
    Institute of Biotechnology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
    Redirecting Cellular Metabolism by Immobilization of Cultured Plant Cells: A Model Study with Coffea arabica1987In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 26, no 5, p. 1431-1434Article in journal (Refereed)
    Abstract [en]

    Suspension-cultured cells of Coffea arabica have been immobilized by entrapment in calcium alginate gels to mimic natural aggregation. The production of methylxanthine alkaloid was increased up to 13-fold by the immobilization. This increased production has been ascribed to organization of the entrapped cells through physicochemical interactions between the polymer (alginate) and the plant cell wall. It has been shown that the metabolic changes induced by the immobilization are reversible. 

  • 102.
    Hamad, Osama A.
    et al.
    Uppsala University.
    Mitroulis, Ioannis
    Tech Univ Dresden, Germany.
    Fromell, Karin
    Uppsala University.
    Kozarcanin, Huda
    Uppsala University.
    Chavakis, Triantafyllos
    Tech Univ Dresden, Germany.
    Ricklin, Daniel
    Univ Penn, USA.
    Lambris, John D.
    Univ Penn, USA.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    Nilsson, Bo
    Uppsala University.
    Contact activation of C3 enables tethering between activated platelets and polymorphonuclear leukocytes via CD11b/CD182015In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 114, no 6, p. 1207-1217Article in journal (Refereed)
    Abstract [en]

    Complement component C3 has a potential role in thrombotic pathologies. It is transformed, without proteolytic cleavage, into C3(H2O) upon binding to the surface of activated platelets. We hypothesise that C3(H2O) bound to activated platelets and to platelet-derived microparticles (PMPs) contributes to platelet-PMN complex (PPC) formation and to the binding of PMPs to PMNs. PAR-1 activation of platelets in human whole blood from normal individuals induced the formation of CD16(+)/CD42a(+) PPC. The complement inhibitor compstatin and a C5a receptor antagonist inhibited PPC formation by 50 %, while monoclonal antibodies to C3(H2O) or anti-CD11b inhibited PPC formation by 75-100 %. Using plasma protein-depleted blood and blood from a C3-deficient patient, we corroborated the dependence on C3, obtaining similar results after reconstitution with purified C3. By analogy with platelets, PMPs isolated from human serum were found to expose C3(H2O) and bind to PMNs. This interaction was also blocked by the anti-C3(H2O) and anti-CD11b monoclonal antibodies, indicating that C3(H2O) and CD11b are involved in tethering PMPs to PMNs. We confirmed the direct interaction between C3(H2O) and CD11b by quartz crystal microbalance analysis using purified native C3 and recombinant CD11b/CD18 and by flow cytometry using PMP and recombinant CD11b. Transfectants expressing CD11b/CD18 were also shown to specifically adhere to surface-bound C3(H2O). We have identified contact-activated C3(H2O) as a novel ligand for CD11b/CD18 that mediates PPC formation and the binding of PMPs to PMNs. Given the various roles of C3 in thrombotic reactions, this finding is likely to have important pathophysiological implications.

  • 103.
    Han, Junli
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Wang, Hongzhen
    Zhejiang Agr & Forestry Univ, Peoples Republic of China.
    Kanagarajan, Selvaraju
    Swedish University of Agricultural Sciences.
    Hao, Mengshu
    Lund University.
    Lundgren, Anneli
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Promoting Artemisinin Biosynthesis in Artemisia annua Plants by Substrate Channeling2016In: Molecular Plant, ISSN 1674-2052, E-ISSN 1752-9867, Vol. 9, no 6, p. 946-948Article in journal (Refereed)
  • 104.
    Han, Junli
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Wang, Hongzhen
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Zhejiang Agriculture and Forestry University, Linan 311300, Zhejiang, PR China..
    Lundgren, Anneli
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Effects of overexpression of AaWRKY1 on artemisinin biosynthesis in transgenic Artemisia annua plants2014In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 102, p. 89-96Article in journal (Refereed)
    Abstract [en]

    The effective anti-malarial medicine artemisinin is costly because of the low content in Artemisia annua. Genetic engineering of A. annua is one of the most promising approaches to improve the yield of artemisinin. In this work, the transcription factor AaWRKY1, which is thought to be involved in the regulation of artemisinin biosynthesis, was cloned from A. annua var. Chongqing and overexpressed using the CaMV35S promoter or the trichome-specific CYP71AV1 promoter in stably transformed A. annua plants. The transcript level of AaWRKY1 was increased more than one hundred times under the CaMV35S promoter and about 40 times under the CYP71AV1 promoter. The overexpressed AaWRKY1 activated the transcription of CYP71AV1 and moreover the trichome-specific overexpression of AaWRKY1 improved the transcription of CYP71AV1 much more effectively than the constitutive overexpression of AaWRKY1, i.e. up to 33 times as compared to the wild-type plant. However the transcription levels of FDS, ADS, and DBR2 did not change significantly in transgenic plants. The significantly up-regulated CYP71AV1 promoted artemisinin biosynthesis, i.e. up to about 1.8 times as compared to the wild-type plant. It is demonstrated that trichome-specific overexpression of AaWRKY1 can significantly activate the transcription of CYP71AV1 and the up-regulated CYP71AV1 promotes artemisinin biosynthesis.

  • 105.
    Harboe, Morten
    et al.
    Oslo University Hospital, Norway.
    Johnson, Christina
    Oslo University Hospital, Norway.
    Nymo, Stig
    Nordland Hospital, Norway.
    Ekholt, Karin
    Oslo University Hospital, Norway.
    Schjalm, Camilla
    Oslo University Hospital, Norway.
    Lindstad, Julie K.
    Oslo University Hospital, Norway.
    Pharo, Anne
    Oslo University Hospital, Norway.
    Hellerud, Bernt Christian
    Oslo University Hospital, Norway.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    Mollnes, Tom Eirik
    Oslo University Hospital, Norway ; Nordland Hospital, Norway ; University of Oslo, Norway ; University of Tromsø, Norway ; Norwegian University of Science and Technology, Norway.
    Nilsson, Per H.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Oslo University Hospital, Norway ; University of Oslo, Norway.
    Properdin binding to complement activating surfaces depends on initial C3b deposition2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 4, p. E534-E539Article in journal (Refereed)
    Abstract [en]

    Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions.

  • 106.
    Haugaard-Kedström, Linda M.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Univ Queensland, Australia.
    Hossain, Mohammed Akhter
    Univ Melbourne, Australia.
    Daly, Norelle L
    Univ Queensland, Australia.
    Bathgate, Ross A D
    Univ Melbourne, Australia.
    Rinderknecht, Ernst
    Novartis Corp, USA.
    Wade, John D
    Univ Melbourne, Australia.
    Craik, David J
    Univ Queensland, Australia.
    Rosengren, K. Johan
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Univ Queensland, Australia.
    Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin-2.2015In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 10, no 3, p. 891-900Article in journal (Refereed)
    Abstract [en]

    Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.

  • 107. Heimgartner, U
    et al.
    Pietrzak, M
    Geertsen, R
    Brodelius, Peter
    Biotechnology ETH-Hönggerberg CH-8093 Zürich, Switzerland .
    da Silva Figueiredo, A C
    S Pais, Maria
    Purification and Partial Characterization of Milk Clotting Proteases from Flowers of Cynara cardunculus1990In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 29, no 5, p. 1405-1410Article in journal (Refereed)
    Abstract [en]

    Three proteases (cynarases 1, 2 and 3) with milk-clotting activity have been purified from dried flowers of Cynara cardunculus. The proteases are each composed of one large and one small subunit. The native Mr of the dimeric proteins is 49 000. The three proteases are glycoproteins containing N-linked high mannose type glycans. Cynarase 3 shows the highest proteolytic and milk-clotting activity. All three enzymes express maximum activity at pH 5.1. Inhibitor studies indicate that the cynarases are of the aspartic acid type. Antibodies raised against the large subunit of cynarase 3 cross-reacts with the large subunits of the other two cynarases after destruction of the glycan structure by periodate oxidation. 

  • 108.
    Henn, Arnon
    et al.
    Technion, Israel.
    Shneyer, Boris
    Technion, Israel.
    Ušaj, Marko
    Technion, Israel.
    Myosin 19 is an Outer Mitochondrial Membrane Motor and Effector of Starvation Induced Filopodia with Unique Kinetic Features2016In: Biophysical Journal Supplement 1, 2016, Vol. 110, p. 615a-616a, article id 3040-PosConference paper (Refereed)
    Abstract [en]

    The interaction between the actin cytoskeleton, myosin motors and their function in mitochondria dynamics, morphology and cellular localization is now beginning to emerge. A novel function for actin-based motors as regulators of cellular adaptations to stress, linking actin cytoskeleton remodelling to mitochondria dynamics. We reveal a novel function for myosin 19 in mitochondrial dynamics and localization during cellular response to glucose starvation. Ectopically expressed myosin 19 localizes with mitochondria at the tips of starvation-induced filopodia. Corollary to this, RNAi mediated knockdown of myosin 19 diminished their formation without evident effects on the mitochondrial network. We analyzed myosin 19 mitochondria interaction and demonstrated that it is uniquely anchored to the outer mitochondrial membrane (OMM) via a 30-residue motif, indicating that myosin 19 is a stably attached OMM molecular motor. To this end, we have purified myosin 19-3IQ motor domain construct. Myosin 19-3IQ featured characteristic actin-activated ATPase activity with moderate to slow turnover (kcat) and relatively tight KATPase. Our transient kinetics and steady state equilibrium binding experiments revealed that myosin 19-3IQ binds ATP and ADP with tight affinity that, to the best of our knowledge, have not yet been exhibited by any other myosins. We suspect that this feature allows myosin 19 to operate in a unique cellular environment that may be related to cellular stress conditions as we showed in our previous studies. The detailed knowledge of myosin 19 enzymatic adaptation will provide us with a quantitative working model of myosin 19, and will assist us to understand its cellular function. Our work reveals a novel function for myosin 19 in mitochondrial positioning during homeostasis and under stress conditions and broadens our understanding of the actin cytoskeleton- myosin -mitochondria interplay.

  • 109. Hulst, A C
    et al.
    Tramper, J
    Brodelius, Peter
    Institute of Biotechnology, Swiss Federal Institute of Technology, Hönggerberg, CH-8093 Zürich, Switzerland.
    Eijkenboom, L J C
    Luyben, K Ch A M
    Immobilized Plant Cells: Respiration and Oxygen Transfer1985In: Journal of chemical technology and biotechnology (1986), ISSN 0268-2575, E-ISSN 1097-4660, Vol. 35, no 3, p. 198-204Article in journal (Refereed)
    Abstract [en]

    The influence of support material (calcium alginate, x-carrageenan and agarose), cell loading and, in case of alginate, bead diameter on the rate of respiration of immobilised plant cells (Daucus carota) was investigated. No significant differences were observed between the three supports and no loss of respiration activity occurred as result of immobilisation per se. The results show further that above a critical combination of cell loading and bead diameter limitations of the rate of respiration by diffusion of oxygen increases with increasing loading and diameter. 

  • 110. Höjeberg, B
    et al.
    Brodelius, Peter
    UNIV LUND, CTR CHEM, DEPT PURE & APPL BIOCHEM, S-22007 LUND 7, SWEDEN .
    Rydström, J
    Mosbach, K
    Affinity Chromatography and Binding Studies on Immobilized Adenosine 5'-Monophosphate and Adenosine 2'.5'-Bisphosphate of Nicotinamide Nucleotide Transhydrogenase from Pseudomonas aeruginosa1976In: European Journal of Biochemistry, Vol. 66, no 3, p. 467-475Article in journal (Refereed)
    Abstract [en]

    Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6-aminoyl)-adenosine-2′,5′-bisphosphate-Sepharose 4B. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 ± 2000. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2′-monophosphate and Ca2+were activators whereas NADP+was inhibitory.  Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2′,5′-bisphosphate-Sepharose and N6-(6-aminohexyl)-adenosine-5′-monophospliate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). Binding of transhydrogenase to N6-(6-aminohexyl)-adenosine-2′,5′-bisphosphate-Sepharose and activation of the enzyme by adenosine-2′,5′-bisphosphate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2′,5′-bisphosphate was virtually constant at various pH values. This discrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites. 

  • 111.
    Israelsson, Stina
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Sävneby, Anna
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ekström, Jens-Ola
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. Umeå universitet.
    Jonsson, Nina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Edman, Kjell
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Lindberg, A. Michael
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Improved replication efficiency of echovirus 5 after transfection of colon cancer cells using an authentic 5' RNA genome end methodology2014In: Investigational new drugs, ISSN 0167-6997, E-ISSN 1573-0646, Vol. 32, no 6, p. 1063-1070Article in journal (Refereed)
    Abstract [en]

    Oncolytic virotherapy is a promising novel form of cancer treatment, but the therapeutic efficiency needs improvement. A potential strategy to enhance the therapeutic effect of oncolytic viruses is to use infectious nucleic acid as therapeutic agent to initiate an oncolytic infection, without administrating infectious viral particles. Here we demonstrate improved viral replication activation efficiency when transfecting cells with 5’ end authentic in vitro transcribed enterovirus RNA as compared to genomic RNA with additional non-genomic 5’ nucleotides generated by conventional cloning methods. We used echovirus 5 (E5) as an oncolytoc model virus due to its ability to replicate in and completely destroy five out of six colon cancer cell lines and kill artificial colon cancer tumors (HT29 spheroids), as shown here. An E5 infectious cDNA clone including a hammerhead ribozyme sequence was used to generate in vitro transcripts with native 5’ genome ends. In HT29 cells, activation of virus replication is approximately 20-fold more efficient for virus genome transcripts with native 5’ genome ends compared to E5 transcripts generated from a standard cDNA clone. This replication advantage remains when viral progeny release starts by cellular lysis 22 h post transfection. Hence, a native 5’ genomic end improves infection activation efficacy of infectious nucleic acid, potentially enhancing its therapeutic effect when used for cancer treatment. The clone design with a hammerhead ribozyme is likely to be applicable to a variety of oncolytic positive sense RNA viruses for the purpose of improving the efficacy of oncolytic virotherapy.

  • 112. Jackson, J B
    et al.
    Lever, T M
    Rydström, J
    Persson, Bengt L.
    Department of Biochemistry and Biotechnology, Royal Institute of Technology .
    Carlenor, E
    Proton-translocating transhydrogenase from photosynthetic bacteria1991In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 19, no 3, p. 573-575Article in journal (Refereed)
  • 113.
    Jacobsson, Erik
    et al.
    Uppsala University.
    Andersson, Håkan S.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Strand, Malin
    Swedish University of Agricultural Sciences.
    Lebbe, Eline
    University of Leuven (KU Leuven), Belgium.
    Eriksson, Camilla
    Uppsala University.
    Peigneur, Steve
    University of Leuven (KU Leuven), Belgium.
    Rosengren, K. Johan
    University of Queensland, Australia.
    Tytgat, Jan
    University of Leuven (KU Leuven), Belgium.
    Göransson, Ulf
    Uppsala University.
    Peptide toxins from the longest animal on earth.2016In: Planta Medica, ISSN 0032-0943, E-ISSN 1439-0221, Vol. 82, no Supplement 1, article id YRW3Article in journal (Other academic)
  • 114.
    Jacobsson, Erik
    et al.
    Uppsala University, Sweden.
    Andersson, Håkan S.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Strand, Malin
    Swedish University of Agricultural Sciences, Sweden.
    Peigneur, Steve
    Univ Leuven, Belgium.
    Eriksson, Camilla
    Uppsala University, Sweden.
    Loden, Henrik
    Uppsala University, Sweden.
    Shariatgorji, Mohammadreza
    Uppsala University, Sweden.
    Andren, Per E.
    Uppsala University, Sweden.
    Lebbe, Eline K. M.
    Univ Leuven, Belgium.
    Rosengren, K. Johan
    Univ Queensland, Australia.
    Tytgat, Jan
    Univ Leuven, Belgium.
    Göransson, Ulf
    Uppsala University, Sweden.
    Peptide ion channel toxins from the bootlace worm, the longest animal on Earth2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4596Article in journal (Refereed)
    Abstract [en]

    Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the alpha-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the 'bootlace worm' known as the longest animal on earth. The most abundant peptide, the 31-residue long alpha-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight alpha-nemertides, mainly distributed in the genus Lineus. alpha-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 mu g/kg (similar to 300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Na(v)1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that a-nemertides can be promising candidates for development of bioinsecticidal agents.

  • 115.
    Johansson, Renzo
    et al.
    Lund University.
    Jonna, Venkateswara Rao
    Umeå University.
    Kumar, Rohit
    Lund University.
    Nayeri, Niloofar
    Lund University.
    Lundin, Daniel
    Stockholm University.
    Sjöberg, Britt-Marie
    Stockholm University.
    Hofer, Anders
    Umeå University.
    Logan, Derek T.
    Lund University.
    Structural Mechanism of Allosteric Activity Regulation in a Ribonucleotide Reductase with Double ATP Cones2016In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 24, no 6, p. 906-917Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides. Their overall activity is stimulated by ATP and downregulated by dATP via a genetically mobile ATP cone domain mediating the formation of oligomeric complexes with varying quaternary structures. The crystal structure and solution X-ray scattering data of a novel dATP-induced homotetramer of the Pseudomonas aeruginosa class I RNR reveal the structural bases for its unique properties, namely one ATP cone that binds two dATP molecules and a second one that is non-functional, binding no nucleotides. Mutations in the observed tetramer interface ablate oligomerization and dATP-induced inhibition but not the ability to bind dATP. Sequence analysis shows that the novel type of ATP cone may be widespread in RNRs. The present study supports a scenario in which diverse mechanisms for allosteric activity regulation are gained and lost through acquisition and evolutionary erosion of different types of ATP cone.

  • 116.
    Jonna, Venkateswara Rao
    et al.
    Umeå University.
    Crona, Mikael
    Stockholm University.
    Rofougaran, Reza
    Umeå University.
    Lundin, Daniel
    Stockholm University.
    Johansson, Samuel
    Umeå University.
    Brännström, Kristoffer
    Umeå University.
    Sjöberg, Britt-Marie
    Stockholm University.
    Hofer, Anders
    Umeå University.
    Diversity in Overall Activity Regulation of Ribonucleotide Reductase2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, p. 1-24Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli canonical class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind one dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.

  • 117.
    Jonsson, Nina
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Sävneby, Anna
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Gullberg, Maria
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Evertsson, Kim
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Klingel, Karin
    University of Tübingen, Germany.
    Lindberg, A. Michael
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Efficient replication of recombinant Enterovirus B types, carrying different P1 genes in the coxsackievirus B5 replicative backbone2015In: Virus genes, ISSN 0920-8569, E-ISSN 1572-994X, Vol. 50, no 3, p. 351-357Article in journal (Refereed)
    Abstract [en]

    Recombination is an important feature in theevolution of the Enterovirus genus. Phylogenetic studies ofenteroviruses have revealed that the capsid genomic region(P1) is type specific, while the parts of the genome codingfor the non-structural proteins (P2–P3) are species specific.Hence, the genome may be regarded as consisting of twomodules that evolve independently. In this study, it wasinvestigated whether the non-structural coding part of thegenome in one type could support replication of a virus witha P1 region from another type of the same species. A cas-sette vector (pCas) containing a full-length cDNA copy ofcoxsackievirus B5 (CVB5) was used as a replicative back-bone. The P1 region of pCas was replaced with the corre-sponding part from coxsackievirus B3Nancy(CVB3N),coxsackievirus B6Schmitt(CVB6S), and echovirus 7Wal-lace(E7W), all members of theEnterovirus Bspecies. Thereplication efficiency after transfection with clone-derivedin vitro transcribed RNA was studied and compared withthat of pCas. All the recombinant viruses replicated with similar efficiencies and showed threshold cycle (Ct) values,tissue culture infectivity dose 50 %, and plaque-forming unittiters comparable to viruses generated from the pCas con-struct. In addition to this, a clone without the P1 region wasalso constructed, and Western Blot and immunofluorescencestaining analysis showed that the viral genome could betranslated and replicated despite the lack of the structuralprotein-coding region. To conclude, the replicative back-bone of the CVB5 cassette vector supports replication ofintraspecies constructs with P1 regions derived from othermembers of theEnterovirus Bspecies. In addition to this,the replicative backbone can be both translated and repli-cated without the presence of a P1 region.

  • 118.
    Jönsson, Boel
    Kemivärlden Biotech.
    Jacobsson, Erik (Contributor)
    Uppsala University.
    Andersson, Håkan S. (Contributor)
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Världens längsta djur gör gift2018In: Kemivärlden Biotech/Kemisk tidskrift, ISSN 1653-5596, no 3, p. 20-21Article in journal (Other (popular science, discussion, etc.))
    Abstract [sv]

    Världens längsta djur, slemmasken långsnöre, producerar nervgifter som kan döda både krabbor och kackerlackor.

  • 119.
    Kanagarajan, Selvaraju
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Tolf, Conny
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Lundgren, Anneli
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Waldenström, Jonas
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, article id e33010Article in journal (Refereed)
    Abstract [en]

    The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6xHis) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6xHis tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.

  • 120.
    Khrennikov, Andrei
    et al.
    Linnaeus University, Faculty of Technology, Department of Mathematics.
    Kozyrev, Sergei
    Russian Acad Sci, Russia.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Hierarchical model of the actomyosin molecular motor based on ultrametric diffusion with drift2015In: Infinite Dimensional Analysis Quantum Probability and Related Topics, ISSN 0219-0257, Vol. 18, no 2, article id 1550013Article in journal (Refereed)
    Abstract [en]

    We discuss the approach to investigate molecular machines using systems of integro-differential ultrametric (p-adic) reaction-diffusion equations with drift. This approach combines the features of continuous and discrete dynamic models. We apply this model to investigation of actomyosin molecular motor. The introduced system of equations is solved analytically using p-adic wavelet theory. We find explicit stationary solutions and behavior in the relaxation regime.

  • 121. Korten, Slobodanka
    et al.
    Albet-Torres, Nuria
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Paderi, Francesca
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Diez, Stefan
    Korten, Till
    te Kronnie, Geertruy
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Sample solution constraints on motor-driven diagnostic nanodevices2013In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 13, no 5, p. 866-876Article in journal (Refereed)
    Abstract [en]

    The last decade has seen appreciable advancements in efforts towards increased portability of lab-on-a-chip devices by substituting microfluidics with molecular motor-based transportation. As of now, first proof-of-principle devices have analyzed protein mixtures of low complexity, such as target protein molecules in buffer solutions optimized for molecular motor performance. However, in a diagnostic workup, lab-on-a-chip devices need to be compatible with complex biological samples. While it has been shown that such samples do not interfere with crucial steps in molecular diagnostics (for example antibody-antigen recognition), their effect on molecular motors is unknown. This critical and long overlooked issue is addressed here. In particular, we studied the effects of blood, cell lysates and solutions containing genomic DNA extracts on actomyosin and kinesin-microtubule-based transport, the two biomolecular motor systems that are most promising for lab-on-a-chip applications. We found that motor function is well preserved at defined dilutions of most of the investigated biological samples and demonstrated a molecular motor-driven label-free blood type test. Our results support the feasibility of molecular-motor driven nanodevices for diagnostic point-of-care applications and also demonstrate important constraints imposed by sample composition and device design that apply both to kinesin-microtubule and actomyosin driven applications.

  • 122.
    Kumar, Saroj
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Milani, Gloria
    University of Padova, Italy.
    Takatsuki, Hideyo
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Lana, Tobia
    University of Padova, Italy.
    Persson, Malin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Frasson, Chiara
    University of Padova, Italy.
    te Kronnie, Geertruy
    University of Padova, Italy.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Sensing protein antigen and microvesicle analytes using high-capacity biopolymer nano-carriers2016In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 3, p. 836-846Article in journal (Refereed)
    Abstract [en]

    Lab-on-a-chip systems with molecular motor driven transport of analytes attached to cytoskeletal filament shuttles (actin filaments, microtubules) circumvent challenges with nanoscale liquid transport. However, the filaments have limited cargo-carrying capacity and limitations either in transportation speed (microtubules) or control over motility direction (actin). To overcome these constraints we here report incorporation of covalently attached antibodies into self-propelled actin bundles (nanocarriers) formed by cross-linking antibody conjugated actin filaments viafascin, a natural actin-bundling protein. We demonstrate high maximum antigen binding activity and propulsion by surface adsorbed myosin motors. Analyte transport capacity is tested using both protein antigens and microvesicles, a novel class of diagnostic markers. Increased incubation concentration with protein antigen in the 0.1–100 nM range (1 min) reduces the fraction of motile bundles and their velocity but maximum transportation capacity of >1 antigen per nm of bundle length is feasible. At sub-nanomolar protein analyte concentration, motility is very well preserved opening for orders of magnitude improved limit of detection using motor driven concentration on nanoscale sensors. Microvesicle-complexing to monoclonal antibodies on the nanocarriers compromises motility but nanocarrier aggregation via microvesicles shows unique potential in label-free detection with the aggregates themselves as non-toxic reporter elements.

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  • 123.
    Kumar, Saroj
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Delhi Technol Univ, India.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Covalent and non-covalent chemical engineering of actin for biotechnological applications2017In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 35, no 7, p. 867-888Article, review/survey (Refereed)
    Abstract [en]

    The cytoskeletal filaments are self-assembled protein polymers with 8-25 nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems andlab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways.

  • 124.
    Kumar, Saroj
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Persson, Malin
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Lard, Mercy
    Lund University.
    Kronnie, Geertruy Te
    University of Padua, Italy.
    Linke, Heiner
    Lund University.
    Månsson, Alf
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, article id e46298Article in journal (Refereed)
    Abstract [en]

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 mu m(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

  • 125.
    Labriere, Christophe
    et al.
    UiT Arctic Univ Norway, Norway;Univ Aberdeen, UK.
    Kondori, Nahid
    University of Gothenburg.
    Caous, Josefin Seth
    RISE Res Inst Sweden.
    Boomgaren, Marc
    UiT Arctic Univ Norway, Norway.
    Sandholm, Kerstin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    Hansen, Jorn H.
    UiT Arctic Univ Norway, Norway.
    Svenson, Johan
    UiT Arctic Univ Norway, Norway;RISE Res Inst Sweden.
    Development and evaluation of cationic amphiphilic antimicrobial 2,5-diketopiperazines2018In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 24, no 7, article id UNSP e3090Article in journal (Refereed)
    Abstract [en]

    Both pathogenic bacteria and fungi are developing resistance to common antimicrobial treatment at an alarming rate. To counteract this development, it is of essence to develop new classes of antimicrobial agents. One such class is antimicrobial peptides, most of which are derived from the innate immune system. In this study, a series of novel 2,5-diketopiperazines were designed, synthesized, and evaluated for their antimicrobial abilities. The compounds were designed to probe the pharmacophore dictated for short linear mimics of antimicrobial cationic peptides, and as such, the compounds contain a range of cationic and hydrophobic functionalities. Several of the prepared compounds displayed high antimicrobial activities toward bacteria and also against human pathogenic fungi. Of particular interest was the high activity toward fungal strains with an inherent increased resistance toward conventional antifungal agents. The most effective compounds displayed inhibition of Candida glabrata and Candida krusei growth at concentrations between 4 and 8 mu g/mL, which is comparable to commercial antifungal agents in use. Structure activity relationship studies revealed a similar dependence on cationic charge and the volume of the hydrophobic bulk as for linear cationic antimicrobial peptides. Finally, the hemolytic activity of selected compounds was evaluated, which revealed a potential to produce active compounds with attenuation of unwanted hemolysis. The findings highlight the potential of cyclic cationic amphiphilic peptidomimetics as a class of promising compounds for the treatment of infections caused by microorganisms with an increased resistance to conventional antimicrobial agents.

  • 126. Lagerstedt, J O
    et al.
    Kruckeberg, A L
    Berden, J
    Persson, Bengt L.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Yeast phosphate transporting System: Regulated Trafficking of the Pho84 Phosphate Transporting Protein2000In: Molecular biology and physiology of water and solute transport: Proceedings of the 3rd International Conference on Molecular Biology and Physiology of Water and Solute transport, held July 1-4, 2000, in Gothenburg, Sweden / [ed] Stefan Hohmann & Søren Nielson, New York: Kluwer Academic Publishers, 2000, p. 405-414Chapter in book (Other academic)
  • 127.
    Lagerstedt, Jens
    et al.
    Växjö University, Faculty of Mathematics/Science/Technology, Institutionen för biovetenskaper och processteknik.
    Zvyagilskaya, Renata
    Pratt, James
    Växjö University, Faculty of Mathematics/Science/Technology, Institutionen för biovetenskaper och processteknik.
    Pattison-Granberg, Johanna
    Växjö University, Faculty of Mathematics/Science/Technology, Institutionen för biovetenskaper och processteknik.
    Kruckeberg, A L
    Berden, J
    Persson, Bengt L.
    Växjö University, Faculty of Mathematics/Science/Technology, Institutionen för biovetenskaper och processteknik.
    Mutagenic and Functional Analysis of the C-terminus of Saccharomyces cerevisiae Pho84 Phosphate Transporter2002In: FEBS letters, Vol. 526, p. 31-37Article in journal (Refereed)
  • 128.
    Lard, Mercy
    et al.
    Lund University.
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Social Sciences, Department of Sport Science.
    Generosi, Johanna
    Lund University.
    Persson, Malin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Linke, Heiner
    Lund University.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Nanowire-Imposed Geometrical Control in Studies of Actomyosin Motor Function2015In: IEEE Transactions on Nanobioscience, ISSN 1536-1241, E-ISSN 1558-2639, Vol. 14, no 3, p. 289-297Article in journal (Refereed)
    Abstract [en]

    Recently, molecular motor gliding assays with actin and myosin from muscle have been realized on semiconductor nanowires coated with Al2O3. This opens for unique nanotechnological applications and novel fundamental studies of actomyosin motor function. Here, we provide a comparison of myosin-driven actin filament motility on Al2O3 to both nitrocellulose and trimethylchlorosilane derivatized surfaces. We also show that actomyosin motility on the less than 200 nm wide tips of arrays of Al2O3-coated nanowires can be used to control the number, and density, of myosin-actin attachment points. Results obtained using nanowire arrays with different inter-wire spacing are consistent with the idea that the actin filament sliding velocity is determined both by the total number and the average density of attached myosin heads along the actin filament. Further, the results are consistent with buckling of long myosin-free segments of the filaments as a factor underlying reduced velocity. On the other hand, the findings do not support a mechanistic role in decreasing velocity, of increased nearest neighbor distance between available myosin heads. Our results open up for more advanced studies that may use nanowire-based structures for fundamental investigations of molecular motors, including the possibility to create a nanowire-templated bottom-up assembly of 3D, muscle-like structures.

  • 129.
    Lard, Mercy
    et al.
    Lund University.
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Kumar, Saroj
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Delhi Technological University, India.
    Persson, Malin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    te Kronnie, G.
    University of Padova, Italy.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Linke, H.
    Lund University.
    Nano-structuring for molecular motor control2015In: Nano-Structures for Optics and Photonics: Optical Strategies for Enhancing Sensing, Imaging, Communication and Energy Conversion / [ed] Baldassare Di Bartolo, John Collins, Luciano Silvestri, Springer, 2015, p. 459-459Conference paper (Refereed)
    Abstract [en]

    The interaction of self-propelled biological molecular-motors and cytoskeletal filaments holds relevance for a variety of applications such as biosensing, drug screening, diagnostics and biocomputation. The use of these systems for lab-on-a-chip biotechnology applications shows potential for replacement of microfluidic flow by active, molecular-motor driven transport of filaments. The ability to control, confine and detect motile objects in such a system is possible by development of nanostructured surfaces for on-chip applications and fundamental studies of molecular-motors. Here we describe the localized detection (Lard et al., Sci Rep 3:1092, 2013) and fast transport of actin filaments by myosin molecular-motors (Lard et al., Biosens Biolectron 48(0):145–152, 2013), inserted within nanostructures, as a method for biocomputation and molecular concentration. These results include extensive myosin driven concentration of actin filaments on a miniaturized detector, of relevance for use of molecular-motors in a diagnostics platform. Also, we discuss the local enhancement of the fluorescence signal of filaments, relevant for use in a biocomputation device where tracking of potentially thousands of motile objects is of primary significance.

  • 130.
    Lard, Mercy
    et al.
    The Nanometer Structure Consortium (nmC@LU), Division of Solid State Physics, Lund University.
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Kumar, Saroj
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Persson, Malin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    te Kronnie, Geertruy
    Department of Women's and Children's Health, University of Padova.
    Linke, Heiner
    The Nanometer Structure Consortium (nmC@LU), Division of Solid State Physics, Lund University.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ultrafast molecular motor driven nanoseparation and biosensing2013In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 48, p. 145-152Article in journal (Refereed)
    Abstract [en]

    Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10 μm2, with a concentration half-time of approximately 40 s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.

  • 131.
    Lard, Mercy
    et al.
    Lund University.
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Linke, Heiner
    Lund University.
    Tracking Actomyosin at Fluorescence Check Points2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, article id 1092Article in journal (Refereed)
    Abstract [en]

    Emerging concepts for on-chip biotechnologies aim to replace microfluidic flow by active, molecular-motor driven transport of cytoskeletal filaments, including applications in bio-simulation, biocomputation, diagnostics, and drug screening. Many of these applications require reliable detection, with minimal data acquisition, of filaments at many, local checkpoints in a device consisting of a potentially complex network of channels that guide filament motion. Here we develop such a detection system using actomyosin motility. Detection points consist of pairs of gold lines running perpendicular to nanochannels that guide motion of fluorescent actin filaments. Fluorescence interference contrast (FLIC) is used to locally enhance the signal at the gold lines. A cross-correlation method is used to suppress errors, allowing reliable detection of single or multiple filaments. Optimal device design parameters are discussed. The results open for automatic read-out of filament count and velocity in high-throughput motility assays, helping establish the viability of active, motor-driven on-chip applications.

  • 132.
    Larsson, Marie C.
    et al.
    Linköping Univ Hosp.
    Lerm, Maria
    Linköping Univ.
    Angeby, Kristian
    Karolinska Univ Hosp ; Univ W Indies, Jamaica.
    Nordvall, Michaela
    Linköping Univ Hosp.
    Jureen, Pontus
    Publ Hlth Agcy Sweden.
    Schön, Thomas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Medicine and Optometry. Linköping Univ ; Kalmar County Hospital.
    A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability2014In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 106, p. 146-150Article in journal (Refereed)
    Abstract [en]

    The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5 days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4 mg/L, 8 mg/L and 2 mg/L, respectively) compared to extracellularly (MIC90: 0.5 mg/L for AMI and EMB; 0.25 mg/L for LEV). The reverse was the case for INH (IC: 0.064 mg/L vs EC: 0.25 mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs. (C) 2014 Elsevier B.V. All rights reserved.

  • 133.
    Lindberg, Frida W.
    et al.
    Lund University.
    Korten, Till
    Tech Univ Dresden, Germany;Max Planck Inst Mol Cell Biol & Genet, Germany.
    Löfstrand, Anette
    Lund University.
    Rahman, Mohammad A.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Graczyk, Mariusz
    Lund University.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Linke, Heiner
    Lund University.
    Maximov, Ivan
    Lund University.
    Design and development of nanoimprint-enabled structures for molecular motor devices2019In: Materials Research Express, E-ISSN 2053-1591, Vol. 6, no 2, article id 025057Article in journal (Refereed)
    Abstract [en]

    Devices based on molecular motor-driven cytoskeletal filaments, e.g., actin filaments, have been developed both for biosensing and biocomputational applications. Commonly, these devices require nanoscaled tracks for guidance of the actin filaments which has limited the patterning technique to electron beam lithography. Thus, large scale systems become intractable to fabricate at a high throughput within a reasonable time-frame. We have studied the possibility to fabricate molecular motor-based devices using the high throughput, high resolution technique of nanoimprint lithography. Molecular motor-based devices require wide open regions (loading zones) to allow filaments to land for later propulsion into the nanoscale tracks. Such open zones are challenging to fabricate using nanoimprint lithography due to the large amount of material displaced in the process. We found that this challenge can be overcome by introducing nanoscaled pillars inside the loading zones, into which material can be displaced during imprint. By optimising the resist thickness, we were able to decrease the amount of material displaced without suffering from insufficient filling of the stamp. Furthermore, simulations suggest that the shape and positioning of the pillars can be used to tailor the overall cytoskeletal filament transportation direction and behaviour. This is a potentially promising design feature for future applications that however, requires further optimisations before experimental realisation.

  • 134.
    Lindberg, Frida W.
    et al.
    Lund University.
    Norrby, Marlene
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rahman, Mohammad A.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Salhotra, Aseem
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Takatsuki, Hideyo
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Jeppesen, Soren
    Lund University.
    Linke, Heiner
    Lund University.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Controlled Surface Silanization for Actin-Myosin and Biocompatibility of New Polymer Resists2018In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, no 30, p. 8777-8784Article in journal (Refereed)
    Abstract [en]

    Molecular motor-based nanodevices require organized cytoskeletal filament guiding along motility-promoting tracks, confined by motility-inhibiting walls. One way to enhance motility quality on the tracks, particularly in terms of filament velocity but also the fraction of motile filaments, is to optimize the surface hydrophobicity. We have investigated the potential to achieve this for the actin myosin II motor system on trimethylchlorosilane (TMCS)-derivatized SiO2 surfaces to be used as channel floors in nanodevices. We have also investigated the ability to supress motility on two new polymer resists, TU7 (for nanoimprint lithography) and CSAR 62 (for electron beam and deep UV lithography), to be used as channel walls. We developed a chemical-vapor deposition tool for silanizing SiO2 surfaces in a controlled environment to achieve different surface hydrophobicities (measured by water contact angle). In contrast to previous work, we were able to fabricate a wide range of contact angles by varying the silanization time and chamber pressure using only one type of silane. This resulted in a significant improvement of the silanization procedure, producing a predictable contact angle on the surface and thereby predictable quality of the heavy meromyosin (HMM)-driven actin motility with regard to velocity. We observed a high degree of correlation between the filament sliding velocity and contact angle in the range 10-86 degrees, expanding the previously studied range. We found that the sliding velocity on TU7 surfaces was superior to that on CSAR 62 surfaces despite similar contact angles. In addition, we were able to suppress the motility on both TU7 and CSAR 62 by plasma oxygen treatment before silanization. These results are discussed in relation to previously proposed surface adsorption mechanisms of HMM and their relationship to the water contact angle. Additionally, the results are considered for the development of actin-myosin based nanodevices with superior performance with respect to actin-myosin functionality.

  • 135. Linsefors, L
    et al.
    Brodelius, Peter
    UNIV LUND, CTR CHEM, S-22007 LUND 7, SWEDEN.
    Immobilization of Plant Protoplasts: Viability Studies1985In: Plant Cell Reports, ISSN 0721-7714, E-ISSN 1432-203X, Vol. 4, no 1, p. 23-27Article in journal (Refereed)
    Abstract [en]

    Protoplasts of Daucus carota Ca68 and Catharanthusroseus have been inmobilized by entrapment in gelformingpolysaccharides (kappa-carrageenan, agarose andalginate). Uniform spherical beads of carrageenan andagarose containing the protoplasts have been preparedby utilizing an inert hydrophobic phase (vegetableoil). The entrapped protoplasts are viable and stabilizedtowards osmotic shock by the polymeric backbone.Standard methods have been used to study the viabilityand integrity of the entrapped protoplasts. Uponincubation in a relatively simple medium the inmobilizedprotoplasts show a much higher viability after14 days as compared to free protoplasts under the sameconditions. The viability of D. carota protoplasts hasalso been monitored by an enzyme activity present inthe cells (digitoxigenin 58-hydroxylase) . 

  • 136.
    Liu, Meng
    et al.
    Shanghai Jiao Tong University, China.
    Shi, Pu
    Shanghai Jiao Tong University, China.
    Fu, Xueqing
    Shanghai Jiao Tong University, China.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Shen, Qian
    Shanghai Jiao Tong University, China.
    Jiang, Weimin
    Shanghai Jiao Tong University, China.
    He, Qian
    Shanghai Jiao Tong University, China.
    Tang, Kexuan
    Shanghai Jiao Tong University, China.
    Characterization of a trichome-specific promoter of the aldehyde dehydrogenase 1 (ALDH1) gene in Artemisia annua2016In: Plant Cell Tissue and Organ Culture, ISSN 0167-6857, E-ISSN 1573-5044, Vol. 126, no 3, p. 469-480Article in journal (Refereed)
    Abstract [en]

    Artemisinin is a frequently used anti-malaria drug extracted from glandular trichomes (GSTs) in Artemisia annua L. In this study, we report on the characterization of the promoter of aldehyde dehydrogenase 1 (ALDH1) involved in the biosynthesis of artemisinin. A 1620-bp promoter fragment was cloned upstream of the ALDH1 start codon. Putative regulatory cis-acting elements are predicted by software, revealing that this gene is affected by complex factors. The activity of the ALDH1 promoter was analyzed using a reporter gene GUS. GUS expression showed a spatial difference in leaves at different ages. In young leaves, GUS staining was exclusively discovered in GSTs. In older leaves, both GSTs and T-shaped trichomes (TSTs) showed GUS signals. Only TSTs showed GUS staining in lower leaves. No GUS staining was detected in the bottom leaves. The result demonstrates that the ALDH1 promoter is trichome-specific. The RT-Q-PCR analysis revealed that both wild-type and recombinant promoters showed similar activity in A. annua. After application of exogenous 100 μM methyl jasmonate, 100 μM gibberellin and 100 μM salicylic acid separately, the transcript levels were increased significantly, indicating that ALDH1 may play an important role in the response to hormones in A. annua.

  • 137.
    Liu, Yu
    et al.
    The Scripps Research Institute, USA.
    Tan, Yun Lei
    The Scripps Research Institute, USA.
    Zhang, Xin
    The Scripps Research Institute, USA.
    Bhabha, Gira
    University of California, USA.
    Ekiert, Damian C
    University of California, USA.
    Genereux, Joseph C
    The Scripps Research Institute, USA.
    Cho, Younhee
    The Scripps Research Institute, USA.
    Kipnis, Yakov
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    Kelly, Jeffery W
    The Scripps Research Institute, USA.
    Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts.2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 12, p. 4449-4454Article in journal (Refereed)
    Abstract [en]

    Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.

  • 138.
    Liu, Yu
    et al.
    The Scripps Research Institute, USA.
    Zhang, Xin
    The Scripps Research Institute, USA.
    Tan, Yun Lei
    The Scripps Research Institute, USA.
    Bhabha, Gira
    University of California, USA.
    Ekiert, Damian C
    University of California, USA.
    Kipnis, Yakov
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    Kelly, Jeffery W
    The Scripps Research Institute, USA.
    De Novo-Designed Enzymes as Small-Molecule-Regulated Fluorescence Imaging Tags and Fluorescent Reporters2014In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, no 38, p. 13102-13105Article in journal (Refereed)
    Abstract [en]

    Enzyme-based tags attached to a protein-of-interest (POI) that react with a small molecule, rendering the conjugate fluorescent, are very useful for studying the POI in living cells. These tags are typically based on endogenous enzymes, so protein engineering is required to ensure that the small-molecule probe does not react with the endogenous enzyme in the cell of interest. Here we demonstrate that de novo-designed enzymes can be used as tags to attach to POIs. The inherent bioorthogonality of the de novo-designed enzyme-small-molecule probe reaction circumvents the need for protein engineering, since these enzyme activities are not present in living organisms. Herein, we transform a family of de novo-designed retroaldolases into variable-molecular-weight tags exhibiting fluorescence imaging, reporter, and electrophoresis applications that are regulated by tailored, reactive small-molecule fluorophores.

  • 139.
    Loderer, Christoph
    et al.
    Stockholm university.
    Jonna, Venkateswara Rao
    Umeå University.
    Crona, Mikael
    Stockholm University.
    Grinberg, Inna Rozman
    Stockholm University.
    Sahlin, Margareta
    Stockholm University.
    Hofer, Anders
    Umeå University.
    Lundin, Daniel
    Stockholm University.
    Sjöberg, Britt-Marie
    Stockholm University.
    A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 46, p. 19044-19054Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.

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  • 140. Lundberg, P
    et al.
    Linsefors, L
    Vogel, H J
    Brodelius, Peter
    Institute of Biotechnology, Swiss Federal Institute of Technology, H6nggerberg, CH-8093 ZOrich, Switzerland.
    Permeabilization of Plant Cells: 31P NMR Studies of the Permeability of the Tonoplast1986In: Plant Cell Reports, ISSN 0721-7714, E-ISSN 1432-203X, Vol. 5, p. 13-16Article in journal (Refereed)
    Abstract [en]

    A suspension culture of Catharanthus roseus has been used to study the permeability of cell membranes after treatment with various concentrations of a permeabilizing agent (DMSO). The uptake and release (after permeabilization) of inorganic phosphate (Pi) by cells have been investigated by 32P radiotracer and non-invasive phosphorus-31 NMR experiments. These studies have demonstrated that measurements of the Pi-efflux from plant cells provide a reliable measure of the permeability of the tonoplast. 

  • 141.
    Lundh, Fredrik
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Mouillon, Jean-Marie
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Samyn, Dieter R.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Stadler, Kent
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Popova, Yulia
    Lagerstedt, Jens
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Thevelein, Johan
    Persson, Bengt L.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Molecular mechanisms controlling phosphate-induced downregulation of the yeast Pho84 phosphate transporter2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 21, p. 4497-4505Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, phosphate uptake is mainly dependent on the proton-coupled Pho84 permease under phosphate-limited growth conditions. Phosphate addition causes Pho84-mediated activation of the protein kinase A (PKA) pathway as well as rapid internalization and vacuolar breakdown of Pho84. We show that Pho84 undergoes phosphate-induced phosphorylation and subsequent ubiquitination on amino acids located in the large middle intracellular loop prior to endocytosis. The attachment of ubiquitin is dependent on the ubiquitin conjugating enzymes Ubc2 and Ubc4. In addition, we show that the Pho84 endocytotic process is delayed in strains with reduced PKA activity. Our results suggest that Pho84-mediated activation of the PKA pathway is responsible for its own downregulation by phosphorylation, ubiquination, internalization, and vacuolar breakdown.

  • 142.
    Maganhi, Stella Hernandez
    et al.
    Federal University of São Carlos, Brazil.
    Jensen, Patrizia
    National Center for Tumor Diseases (NCT), Germany;German Cancer Research Center (DKFZ) Heidelberg, Germany.
    Caracelli, Ignez
    Federal University of São Carlos, Brazil.
    Schpector, Julio Zukerman
    Federal University of São Carlos, Brazil.
    Froehling, Stefan
    National Center for Tumor Diseases (NCT), Germany;German Cancer Research Center (DKFZ) Heidelberg, Germany ; Heidelberg University Hospital, Germany ; German Cancer Consortium (DKTK), Germany.
    Friedman, Ran
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Palbociclib can overcome mutations in cyclin dependent kinase 6 that break hydrogen bonds between the drug and the protein2017In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 26, no 4, p. 870-879Article in journal (Refereed)
    Abstract [en]

    Inhibition of cyclin dependent kinases (CDKs) 4 and 6 prevent cells from entering the synthesis phase of the cell cycle. CDK4 and 6 are therefore important drug targets in various cancers. The selective CDK4/6 inhibitor palbociclib is approved for the treatment of breast cancer and has shown activity in a cellular model of mixed lineage leukaemia (MLL)-rearranged acute myeloid leukaemia (AML). We studied the interactions of palbociclib and CDK6 using molecular dynamics simulations. Analysis of the simulations suggested several interactions that stabilized the drug in its binding site and that were not observed in the crystal structure of the protein-drug complex. These included a hydrogen bond to His 100 that was hitherto not reported and several hydrophobic contacts. Evolutionary-based bioinformatic analysis was used to suggest two mutants, D163G and H100L that would potentially yield drug resistance, as they lead to loss of important protein-drug interactions without hindering the viability of the protein. One of the mutants involved a change in the glycine of the well-conserved DFG motif of the kinase. Interestingly, CDK6-dependent human AML cells stably expressing either mutant retained sensitivity to palbociclib, indicating that the protein-drug interactions are not affected by these. Furthermore, the cells were proliferative in the absence of palbociclib, indicating that the Asp to Gly mutation in the DFG motif did not interfere with the catalytic activity of the protein.

  • 143.
    Mandal, Sudip
    et al.
    Indian Inst Technol Madras, India.
    Suriyanarayanan, Subramanian
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Nicholls, Ian A.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ramanujam, Kothandaraman
    Indian Inst Technol Madras, India.
    Selective Sensing of the Biotinyl Moiety Using Molecularly Imprinted Polyaniline Nanowires2018In: Journal of the Electrochemical Society, ISSN 0013-4651, E-ISSN 1945-7111, Vol. 165, no 14, p. B669-B678Article in journal (Refereed)
    Abstract [en]

    A chemosensor for selective recognition of biotinyl moiety has been devised using electropolymerized film and tested against selective biotinylated targets. The sensor comprises biotin molecularly imprinted polymer (MIP) polymeric nanowires, as a recognition element, overlaid on gold-coated quartz transducers. The preparation of nanostructured MIPs and reference systems have been demonstrated using electrochemical copolymerization of the stabilized complex between the template (biotin), the functional monomer (4-aminobenzoic acid), and cross-linker (aniline) and/or sacrificial biotin-modified Al2O3 membrane. Density functional theoretical studies signify formation of a stable hydrogen-bonded complex of biotin with 4-aminobenzoic acid in the pre-polymerization mixture. Scanning electron microscope studies revealed uniformly grown and densely packed polyaniline hierarchical structures. Piezoelectric microgravimetry under flow injection analysis (FIA) conditions revealed selective binding of biotin methyl ester (BtOMe, 4) (79.89 +/- 2.17 Hz/mM) with imprinted polyaniline hierarchical structures over 10 fold higher than the non-imprinted counterpart. The detection limit of the MIP is 50 nM under optimized conditions. Particularly, the sensor selectively recognizes BtOMe from structural or functional analogues, such as thiamine (4.87 +/- 0.10 Hz/mM) and pyridoxamine (12.08 +/- 0.24 Hz/mM). Importantly, the MIP hierarchical structures were shown to be selective for biotinylated targets (biotin moiety labeled cytochrome C, dextran, oxytocin and obestatin). (C) 2018 The Electrochemical Society.

  • 144. Marques, I
    et al.
    Brodelius, Peter
    Institute ofBiotechnology, ETH Hönggerberg, CH-8093 Zurich, Switzerland.
    Elicitor-Induced L-Tyrosine Decarboxylase from Plant Cell Suspension Cultures: II. Partial Characterization :  1988In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 88, p. 52-55Article in journal (Refereed)
    Abstract [en]

    Properties of purified L-tyrosine decarboxylase (EC 4.1.1.25) fromelicitor-induced cell suspension cultures of Eschscholtzia californicaCham. and Thalictrum rugosum Ait. are described. L-Tyrosine decarboxylaseis a dimeric enzyme with a molecular weight of 112,600 ± 600daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4for the enzyme from E. californica and T. rugosum, respectively. Thepurified enzymes were stabilized in the presence of pyridoxal-5-phosphate.Optimum pH for the enzyme from both plants was found to be8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5-phosphate. The enzyme decarboxylated L-tyrosine and L-,a-3,4-dihydroxyphenylalanine but was inactive toward L-phenylalanine and Ltryptophan.Apparent Km values of Eschscholtzia- and Thalictrum-decarboxylasefor L-tyrosine were 0.25 ± 0.03 and 0.27 ± 0.04 millimolar,respectively. Similar affinities were found for L-3,4-dihydroxyphenylalanine.Eschscholtzia L-tyrosine decarboxylase was strongly inhibitedby the phenylalanine analogue L-a-aminooxy-#-phenylpropionate andlargely unaffected by D,L-a-monofluoromethyl-3,4-dihydroxyphenylalanineand a-difluoromethyltyrosine. 

  • 145. Marques, I
    et al.
    Brodelius, Peter
    Institute of Biotechnology, ETH-H6nggerberg, CH-8093 Ztirich, Switzerland.
    Elicitor-induced Tyrosine Decarboxylase from Plant Cell Suspension Cultures. Part I. Induction and Purification1988In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 88, p. 46-51Article in journal (Refereed)
    Abstract [en]

    L-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cellsuspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californicaCham. with a yeast polysaccharide preparation (elicitor). Thehighest L-tyrosine decarboxylase activity in extracts from 7-day-old cellcultures of E. californica was observed 5 hours after addition of 30 to 40micrograms elicitor per gram cell fresh weight. The enzyme extractedfrom cells of E. californica was purified 1540-fold to a specific activityof 2.6 micromoles CO2 produced per minute per millim protein at pH8A and 30°C. Purified enzyme from T. rugosum showed a specific activityof 0.18 micromoles per minute per milligram protein. The purificationprocedure involved ammonium sulfate fractionation, anion-exchange fastprotein liquid chromatography, ultrafiltration, and hydrophobic interactionchromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the enzyme from the two plant cell cultures hadsubunits of identical molecular weight (56,300 1 300 daltons. 

  • 146.
    Matias-Hernandez, Luis
    et al.
    CSIC IRTA UAB UB, Spain ; Sequentia Biotech, Spain.
    Jiang, Weimin
    Shanghai Jiao Tong Univ, Peoples Republic of China.
    Yang, Ke
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Tang, Kexuan
    Shanghai Jiao Tong Univ, Peoples Republic of China.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Pelaz, Soraya
    CSIC IRTA UAB UB, Spain ; ICREA, Spain.
    AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 3, p. 520-534Article in journal (Refereed)
    Abstract [en]

    The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA(9) into the bioactive GA(4) as well as the enzymes involved in the degradation of GA(4). Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.

  • 147.
    Matusovsky, Oleg S.
    et al.
    McGill Univ, Canada.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Persson, Malin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. McGill Univ, Canada.
    Cheng, Yu-Shu
    McGill Univ, Canada.
    Rassier, Dilson E.
    McGill Univ, Canada.
    High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding2019In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 33, p. 16384-16393Article in journal (Refereed)
    Abstract [en]

    High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropo-myosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

  • 148.
    Meiby, Elinor
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Progress of Weak Affinity Chromatography as a Tool in Drug Development2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Weak Affinity Chromatography (WAC) is a technology that was developed to analyse weak (KD > 10-5 M) although selective interactions between biomolecules. The focus of this thesis was to develop this method for various applications in the drug development process.

     

    Fragment Based Drug Discovery is a new approach in finding new small molecular drugs. Here, relatively small libraries (a few hundreds to a few thousands of compounds) of fragments (150 – 300 Da) are screened against the target. Fragment hits are then developed into lead molecules by linking, growing or merging fragments binding to different locations of the protein’s active site. However, due to the weakly binding nature of fragments, methods that are able to detect very weak binding events are needed. In this thesis, WAC is presented as a new robust and highly reproducible technology for fragment screening. The technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein-protein interaction (PPI) targets. Comparison of data from fragment screening of 111 fragments by WAC and other more established technologies for fragment screening, such as surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR), validates WAC as a screening technology. It also points at the importance of performing fragment screening by multiple methods as they complement each other.

     

    Other applications of WAC in drug development are also presented. The method can be used for chiral separations of racemic mixtures during fragment screening, which enables affinity measurements of individual enantiomers binding to the target of interest. Further, analysis of crude reaction mixtures is shown. By these procedures, the affinity of the product can be assessed directly after synthesis without any time-consuming purification steps. In addition, a high performance liquid chromatography (HPLC) system for highly efficient drug partition studies was developed by stable immobilization of lipid bilayer disks – lipodisks – on a high performance silica support material. These lipodisks are recognized model membranes for drug partition studies. A WAC system with incorporated membrane proteins into immobilized lipodisks has also been produced and evaluated with the ultimate objective to study affinity interactions between ligands and membrane proteins.

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  • 149.
    Meiby, Elinor
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Knapp, Stefan
    Elkins, Jonathan M.
    Ohlson, Sten
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Fragment screening of cyclin G-associated kinase by weak affinity chromatography2012In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, no 8, p. 2417-2425Article in journal (Refereed)
    Abstract [en]

    Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K (D)) = mM -aEuro parts per thousand mu M) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K (D) < 200 mu M were detected, including a few highly efficient GAK binders (K (D) of 2 mu M; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

  • 150.
    Meiby, Elinor
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    M Zetterberg, Malin
    Uppsala University.
    Victor, Hernàndez
    Uppsala University.
    Ohlson, Sten
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore.
    Edwards, Katarina
    Uppsala University.
    Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis.2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 14, p. 4859-4869Article in journal (Refereed)
    Abstract [en]

    Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.

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