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  • 151. Mobley, K. B.
    et al.
    Amundsen, T.
    Forsgren, E.
    Svensson, P. Andreas
    School of Biological Sciences, Monash University, Melbourne, VIC 3800, Australia.
    Jones, A.G.
    Multiple mating and a low incidence of cuckoldry for nest-holding males in the two-spotted goby, Gobiusculus flavescens2009In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 9, p. 1-10, article id 6Article in journal (Refereed)
    Abstract [en]

    Background: A major question in behavioural ecology concerns the relationship between genetic mating systems and the strength of sexual selection. In this study, we investigated the genetic mating system of the two-spotted goby (Gobiusculus flavescens), a useful fish model for the study of sexual selection whose genetic mating system remains uncharacterized. We developed four polymorphic microsatellite markers and used them to conduct parentage analyses on 21 nests collected during the breeding season to examine the rates of multiple mating by males and to test for evidence of alternative mating strategies. Results: Results of this study indicate that male G. flavescens mate with multiple females and enjoy confidence of paternity. We detected only one instance of sneaking, so cuckoldry contributed a very small percentage (~0.1%) of the total fertilizations in this population. Nests were nearly full and males that maintain larger nests have higher mating and reproductive success, irrespective of body size. Conclusion: Overall, our investigation shows that G. flavescens is similar to other, related gobies in that the nests of care-giving males often contain eggs from multiple females. However, G. flavescens differs from other gobies in displaying an extremely low rate of cuckoldry. The study of ecological factors responsible for this important difference between G. flavescens and related species should be a fertile area for future work.

  • 152.
    Mohlin, Camilla
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Delbro, Dick
    Örebro University.
    Kvanta, Anders
    Karolinska Institutet.
    Johansson, Kjell
    Kristianstad University.
    Evaluation of Congo Red Staining in Degenerating Porcine Photoreceptors In Vitro: Protective Effects by Structural and Trophic Support2018In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 66, no 9Article in journal (Refereed)
    Abstract [en]

    Congo red (CR) is a histological stain used for the detection of extracellular amyloids mediating various neurodegenerative diseases. Given that damaged photoreceptors appear to degenerate similarly to other nerve cells, CR staining was evaluated in experimentally injured porcine retina. CR staining appeared mostly as discrete cytosolic deposits with no obvious plaque formation during the investigated time period. Increases of CR labeling coincided temporally with the known accumulation of mislocalized opsins and increases of cell death. Coculture, either with human retinal pigment epithelium (ARPE) or human neural progenitor (ReN) cells, was accompanied by a significant reduction of CR labeling. Of particular interest was the reduction of CR labeling in cone photoreceptors, which are important for the perception of color and fine details and afflicted in age-related macular degeneration (AMD). Electron microscopy revealed inclusions in the inner segment, cell body, and occasionally synaptic terminals of photoreceptor cells in cultured specimens. Closer examinations indicated the presence of different types of inclusions resembling protein aggregates as well as inclusion bodies. The current results indicate that injury-related response resulted in accumulation of CR deposits in photoreceptor cells, and that trophic and/or structural support attenuated this response.

  • 153.
    Momeni, Naghi
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Bergquist, Jonas
    Uppsala University.
    Brudin, Lars
    Kalmar County Hospital.
    Behnia, Fatemeh
    University of Social Welfare and Rehabilitation Sciences, Iran.
    Sivberg, Bengt
    Lund University.
    Joghataei, Mohammad
    Tehran Medical University, Iran.
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    A novel blood-based biomarker for detection of autism spectrum disorders2012In: Translational Psychiatry, ISSN 2158-3188, E-ISSN 2158-3188, Vol. 2, article id e91Article in journal (Refereed)
    Abstract [en]

    Autism Spectrum Disorders (ASD) are classified as neurological developmental disorders. Several studies have been carried out to find a candidate biomarker linked to development of these disorders, but up to date no reliable biomarker is available. Mass spectrometry techniques have been used for protein profiling of blood plasma of children with such disorders in order to identify proteins/peptides which may be used as biomarkers for detection of the disorders. Three differentially expressed peptides with mass charged (m/z) values of 2,020 ± 1, 1,864 ± 1, and 1,978 ± 1 Da in heparin plasma of children with ASD which were significantly changed as compared to the peptide pattern of the non-ASD control group are reported here. This novel set of biomarkers allows for a reliable blood based diagnostic tool that may be used in diagnosis and potentially, in prognosis of ASD. 

  • 154.
    Moretto, Luisa
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Heylen, Rachel
    Univ Cambridge, UK.
    Holroyd, Natalie
    UCL, UK.
    Vance, Steven
    Crescendo Biol Ltd, UK.
    Broadhurst, R. William
    Univ Cambridge, UK.
    Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 2325Article in journal (Refereed)
    Abstract [en]

    Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional alpha-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be similar to 12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.

  • 155.
    Muthusamy, Sarala Devi
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Vetukuri, Ramesh R.
    Swedish university of agricultural sciences, Sweden.
    Lundgren, Anneli
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ganji, Suresh
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Zhu, Li-Hua
    Swedish university of agricultural sciences, Sweden.
    Brodelius, Peter E.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Kanagarajan, Selvaraju
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Swedish Univ Agr Sci, Dept Plant Breeding, Alnarp, Sweden..
    Transient expression and purification of β-caryophyllene synthase in Nicotiana benthamiana to produce β-caryophyllene in vitro2020In: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 8, p. 1-21, article id e8904Article in journal (Refereed)
    Abstract [en]

    The sesquiterpene beta-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, beta-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of beta-caryophyllene. In this study, a full-length cDNA of a new duplicated beta-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce beta-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg(-1). fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce p-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 +/- 1 mg of P-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

  • 156.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Comparing models with one versus multiple myosin-binding sites per actin target zone: The power of simplicity2019In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 151, no 4, p. 578-592Article in journal (Refereed)
    Abstract [en]

    Mechanokinetic statistical models describe the mechanisms of muscle contraction on the basis of the average behavior of a large ensemble of actin-myosin motors. Such models often assume that myosin II motor domains bind to regularly spaced, discrete target zones along the actin-based thin filaments and develop force in a series of strain-dependent transitions under the turnover of ATP. The simplest models assume that there is just one myosin-binding site per target zone and a uniform spatial distribution of the myosin motor domains in relation to each site. However, most of the recently developed models assume three myosin-binding sites per target zone, and some models include a spatially explicit 3-D treatment of the myofilament lattice and thereby of the geometry of the actin-myosin contact points. Here, I show that the predictions for steady-state contractile behavior of muscle are very similar whether one or three myosin-binding sites per target zone is assumed, provided that the model responses are appropriately scaled to the number of sites. Comparison of the model predictions for isometrically contracting mammalian muscle cells suggests that each target zone contains three or more myosin-binding sites. Finally, I discuss the strengths and weaknesses of one-site spatially inexplicit models in relation to three-site models, including those that take into account the detailed 3-D geometry of the myofilament lattice. The results of this study suggest that single-site models, with reduced computational cost compared with multisite models, are useful for several purposes, e.g., facilitated molecular mechanistic insights.

  • 157.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    From Contractile Non-Uniformities and Mechanical Instabilities to Hypertrophic Cardiomyopathy2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2 Supplement 1, p. 444A-444A, article id 2234-PosArticle in journal (Other academic)
  • 158.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    The effects of inorganic phosphate on muscle force development and energetics: challenges in modelling related to experimental uncertainties2019In: Journal of Muscle Research and Cell Motility, ISSN 0142-4319, E-ISSN 1573-2657Article in journal (Refereed)
    Abstract [en]

    Muscle force and power are developed by myosin cross-bridges, which cyclically attach to actin, undergo a force-generating transition and detach under turnover of ATP. The force-generating transition is intimately associated with release of inorganic phosphate (Pi) but the exact sequence of events in relation to the actual Pi release step is controversial. Details of this process are reflected in the relationships between [Pi] and the developed force and shortening velocity. In order to account for these relationships, models have proposed branched kinetic pathways or loose coupling between biochemical and force-generating transitions. A key hypothesis underlying the present study is that such complexities are not required to explain changes in the force-velocity relationship and ATP turnover rate with altered [Pi]. We therefore set out to test if models without branched kinetic paths and Pi-release occurring before the main force-generating transition can account for effects of varied [Pi] (0.1-25 mM). The models tested, one assuming either linear or non-linear cross-bridge elasticity, account well for critical aspects of muscle contraction at 0.5 mM Pi but their capacity to account for the maximum power output vary. We find that the models, within experimental uncertainties, account for the relationship between [Pi] and isometric force as well as between [Pi] and the velocity of shortening at low loads. However, in apparent contradiction with available experimental findings, the tested models produce an anomalous force-velocity relationship at elevated [Pi] and high loads with more than one possible velocity for a given load. Nevertheless, considering experimental uncertainties and effects of sarcomere non-uniformities, these discrepancies are insufficient to refute the tested models in favour of more complex alternatives.

  • 159.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rassier, Dilson
    McGill University, Canada.
    Tsiavaliaris, Georgios
    Hannover Medical School, Germany.
    Poorly Understood Aspects of Striated Muscle Contraction2015In: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, article id 245154Article, review/survey (Refereed)
    Abstract [en]

    Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP). Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs.

  • 160.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    ten Siethoff, Lasse
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Lard, Mercy
    Lund Univ.
    Generosi, Johanna
    Lund Univ.
    Andersson, Håkan S.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Linke, Heiner
    Lund Univ.
    Three-Dimensionally Constrained Actomyosin Motility on Oxide Coated Semiconductor Nanowires2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 453A-453AArticle in journal (Other academic)
  • 161.
    Månsson, Alf
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ušaj, Marko
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Moretto, Luisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rassier, Dilson E.
    McGill Univ, Canada.
    Do Actomyosin Single-Molecule Mechanics Data Predict Mechanics of Contracting Muscle?2018In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 19, no 7, article id 1863Article, review/survey (Refereed)
    Abstract [en]

    In muscle, but not in single-molecule mechanics studies, actin, myosin and accessory proteins are incorporated into a highly ordered myofilament lattice. In view of this difference we compare results from single-molecule studies and muscle mechanics and analyze to what degree data from the two types of studies agree with each other. There is reasonable correspondence in estimates of the cross-bridge power-stroke distance (7-13 nm), cross-bridge stiffness (similar to 2 pN/nm) and average isometric force per cross-bridge (6-9 pN). Furthermore, models defined on the basis of single-molecule mechanics and solution biochemistry give good fits to experimental data from muscle. This suggests that the ordered myofilament lattice, accessory proteins and emergent effects of the sarcomere organization have only minor modulatory roles. However, such factors may be of greater importance under e.g., disease conditions. We also identify areas where single-molecule and muscle data are conflicting: (1) whether force generation is an Eyring or Kramers process with just one major power-stroke or several sub-strokes; (2) whether the myofilaments and the cross-bridges have Hookean or non-linear elasticity; (3) if individual myosin heads slip between actin sites under certain conditions, e.g.,in lengthening; or (4) if the two heads of myosin cooperate.

  • 162.
    Nicholls, Ian A.
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Karlsson, Björn C. G.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Rosengren, Annika M.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Henschel, Henning
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Warfarin: an environment-dependent switchable molecular probe2010In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 23, no 6, p. 604-608Article in journal (Refereed)
    Abstract [en]

    The complex nature of the structure of the anticoagulant warfarin is reflected in the diversity of binding modes observed in warfarin–protein recognition systems. A series of theoretical, 1H-NMR and steady state and time resolved fluorescence spectroscopic studies, have been used to establish correlations between the molecular environment provided by various solvent systems and the relative concentrations of the various members of warfarin's ensemble of isomers. A consequence of these observations is that the judicious choice of solvent system or molecular environment of warfarin allows for manipulation of the position of the equilibrium between isomeric structures such as the hemiacetal and open phenol-keto forms, the latter even possible in a deprotonated form, where in each case unique spectroscopic properties are exhibited by the respective structures. Collectively, warfarin's capacity to adapt its structure as a function of environment in conjunction with the fluorescence behaviours of the various isomers together provide an environment-dependent molecular switch with reporter properties, which allows for the simultaneous detection of warfarin in different states with lifetimes spanning the range < 0.10–5.5 ns. These characteristics are here used to examine warfarin binding domains in a series of materials (solvents, protein, inorganic matrix and synthetic polymer). Moreover, these studies demonstrate the potential for using warfarin, or other switchable analogues thereof, as a tool for studying molecular level characteristics, for example local dielectricity. Copyright © 2010 John Wiley & Sons, Ltd.

  • 163.
    Nilsson, Bo
    et al.
    Uppsala University.
    Teramura, Yuji
    Uppsala University;University of Tokyo.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    The role and regulation of complement activation as part of the thromboinflammation elicited in cell therapies2014In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 61, no 2, p. 185-190Article, review/survey (Refereed)
    Abstract [en]

    Cell therapies in which the cells come into direct contact with blood and other body fluids are emerging treatment procedures for patients with various diseases, such as diabetes mellitus, liver insufficiency, and graft-versus-host disease. However, despite recent progress, these procedures are associated with tissue loss caused by thromboinflammatory reactions. These deleterious reactions involve the activation of the complement and coagulation cascades and platelet and leukocyte activation, ultimately resulting in clot formation and damage to the implanted cells. In this concept review, we discuss the basic mechanisms underlying the thrombininflammatory process, with special reference to the engagement of complement and emerging strategies for the therapeutic regulation of these reactions that include the use of selective systemic inhibitors and various procedures to coat the surfaces of the cells. The coating procedures may also be applied to other treatment modalities in which similar mechanisms are involved, including whole organ transplantation, treatment with biomaterials in contact with blood, and extracorporeal procedures. (C) 2014 Published by Elsevier Ltd.

  • 164.
    Nilsson Ekdahl, Kristina
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Engberg, Anna E.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Rosengren-Holmberg, Jenny P.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Chen, H
    University of Pennsylvania.
    Lambris, JD
    University of Pennsylvania.
    Nicholls, Ian A.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Blood protein-polymer adsorption fingerprinting: Implications for understanding hemoocompatibility and for biomaterial design.2011In: Journal of Biomedical Materials Research. Part A, ISSN 1549-3296, E-ISSN 1552-4965, Vol. 97A, no 1, p. 74-84Article in journal (Refereed)
  • 165.
    Nilsson Ekdahl, Kristina
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    Hong, Jaan
    Uppsala University.
    Hamad, Osama
    Uppsala University.
    Larsson, Rolf
    Uppsala University.
    Nilsson, Bo
    Uppsala University.
    Evaluation of the blood compatibility of materials, cells and tissues: Basic concepts, test models and practical guidelines2013In: Complement Therapeutics / [ed] J.D. Lambris, V.M. Holers & D. Ricklin, Springer, 2013, p. 257-270Chapter in book (Refereed)
    Abstract [en]

    Medicine today uses a wide range of biomaterials, most of which make contact with blood permanently or transiently upon implantation. Contact between blood and nonbiological materials or cells or tissue of nonhematologic origin initiates activation of the cascade systems (complement, contact activation/coagulation) of the blood, which induces platelet and leukocyte activation.

    Although substantial progress regarding biocompatibility has been made, many materials and medical treatment procedures are still associated with severe side effects. Therefore, there is a great need for adequate models and guidelines for evaluating the blood compatibility of biomaterials. Due to the substantial amount of cross talk between the different cascade systems and cell populations in the blood, it is advisable to use an intact system for evaluation.

    Here, we describe three such in vitro models for the evaluation of the biocompatibility of materials and therapeutic cells and tissues. The use of different anticoagulants and specific inhibitors in order to be able to dissect interactions between the different cascade systems and cells of the blood is discussed. In addition, we describe two clinically relevant medical treatment modalities, the integration of titanium implants and transplantation of islets of Langerhans to patients with type 1 diabetes, whose mechanisms of action we have addressed using these in vitro models.

  • 166. Nilsson, K
    et al.
    Birnbaum, S
    Flygare, S
    Linse, L
    Schröder, U
    Jeppsson, U
    Larsson, P-O
    Mosbach, K
    Brodelius, Peter
    Lunds Universitet.
    A General Method for the Immobilization of Cells with Preserved Viability1983In: Applied Microbiology and Biotechnology, Vol. 17, p. 319-326Article in journal (Refereed)
    Abstract [en]

    Microbial, algal, plant and animal cells have been immobilized, with preserved viability, by entrapment in various matrices according to a new bead polymerization technique. The cell polymer/monomer mixture is kept suspended in a hydrophobic phase such as soy, paraffin, or silicon oil, tri-n-butylphosphate, or dibutyphtalate, which is compatible with the cells. The various monomers or polymers tested include agarose, agar, carrageenan, alginate, fibrin, and polyacrylamide. Furthermore, by adjustment of the stirring speed of the suspension, beads of desired diameter can easily be obtained. The entrapped cells are fully viable and biosynthetically active. 

  • 167.
    Nilsson, Per H.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Interactions between platelets and complement with implications for the regulation at surfaces2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces.   

    Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H.

    Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood.

    In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering platelets, which highlights the importance of regulating both complement and platelets to lower inflammatory events. In addition, a strategy to enhance the biocompatibility of biomaterials and cells by simultaneously targeting ADP-dependent platelet activation and the alternative complement C3-convertase is proposed.

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  • 168.
    Nilsson, UR
    et al.
    Uppsala University.
    Funke, Lillemor
    Uppsala University.
    Nilsson, Bo
    Uppsala University.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Two conformational forms of target bound iC3b which distinctively bind complement receptors 1 and 2 (CR1 and CR2) and two specific monoclonal antibodies.2010In: Upsala Journal of Medical Sciences, Supplement, ISSN 0300-9726Article in journal (Other academic)
    Abstract [en]

    Introduction. The complement system is an essential part of the immune system of vertebrates. The central event of thecomplement activation cascade is the sequential proteolytic activation of C3, which is associated with profound alterations inthe molecule’s structure and conformation and is responsible for triggering most of the biological effects of complement.Material and methods. Here, we have studied the conformation of C3 fragments deposited onto an IgG-coated surface fromhuman serum during complement activation, using a set of unique monoclonal antibodies (mAbs) that are all specific for theC3dg portion of bound iC3b.Results. We were able to identify two conformational forms of target-bound iC3b: the first recognized by mAb 7D18.1, and thesecond by mAb 7D323.1. The first species of iC3b bound recombinant complement receptor 1 (CR1), while the second boundCR2. Since CR1 and CR2 are expressed by different subsets of leukocytes, this difference in receptor-binding capacity impliesthat there is a biological difference between the two forms of surface-bound iC3b.Conclusion. We propose that mAbs 7D18.1 and 7D323.1 can act as surrogate markers for CR1 and CR2, respectively, and thatthey may be useful tools for studying the immune complexes that are generated in various autoimmune diseases.

  • 169.
    Nivón, Lucas G
    et al.
    University of Washington, USA.
    Bjelic, Sinisa
    University of Washington, USA.
    King, Chris
    University of Washington, USA.
    Baker, David
    University of Washington, USA.
    Automating human intuition for protein design2014In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, no 5, p. 858-866Article in journal (Refereed)
    Abstract [en]

    In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins.

  • 170.
    Nix, W Allan
    et al.
    Centers for Disease Control and Prevention, Atlanta, Georgia .
    Maher, Kaija
    Centers for Disease Control and Prevention, Atlanta, Georgia .
    Johansson, E Susanne
    University of Kalmar.
    Niklasson, Bo
    Apodemus AB, Stockholm.
    Lindberg, A Michael
    University of Kalmar.
    Pallansch, Mark A
    Centers for Disease Control and Prevention, Atlanta, Georgia .
    Oberste, M Steven
    Centers for Disease Control and Prevention, Atlanta, Georgia .
    Detection of all known parechoviruses by real-time PCR.2008In: Journal of clinical microbiology, ISSN 1098-660X, Vol. 46, no 8, p. 2519-24Article in journal (Refereed)
    Abstract [en]

    The Parechovirus genus of the Picornaviridae family contains two species, Human parechovirus (HPeV) and Ljungan virus (LV). The HPeVs (including the former echoviruses 22 and 23, now HPeV type 1 (HPeV1) and HPeV2, respectively) cause a wide spectrum of disease, including aseptic meningitis, gastroenteritis, encephalitis, acute respiratory illness, and neonatal sepsis-like disease. The LVs were isolated from bank voles in Sweden during a search for an infectious agent linked to fatal myocarditis cases in humans. Because of the decline in use of cell culture and neutralization to investigate enterovirus-like disease, very few laboratories currently have the capability to test for parechoviruses. We have developed a real-time reverse transcription-PCR (RT-PCR) assay for detection of all known members of the genus Parechovirus. The assay targets the conserved regions in the 5' nontranslated region (5'NTR) of the parechovirus genome and can detect both HPeVs and LVs, unlike other published parechovirus 5' NTR assays, which only detect known HPeVs or only LVs. HPeV and LV can be differentiated by sequencing the 5'NTR real-time RT-PCR amplicon, when needed. The assay is approximately 100 times more sensitive than cell culture and may be used to test original clinical specimens. The availability of a broad-specificity PCR method should facilitate the detection of new human parechoviruses, as well as new parechoviruses in other mammalian species, and provide an opportunity to investigate the role of these viruses in human and animal disease.

  • 171.
    Noiri, Makoto
    et al.
    Univ Tokyo, Japan.
    Asawa, Kenta
    Univ Tokyo, Japan.
    Okada, Naoya
    Univ Tokyo, Japan.
    Kodama, Tomonobu
    Jikei Univ Hosp, Japan.
    Murayama, Yuichi
    Jikei Univ Hosp, Japan.
    Inoue, Yuuki
    Univ Tokyo, Japan.
    Ishihara, Kazuhiko
    Univ Tokyo, Japan.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University, Sweden.
    Nilsson, Bo
    Uppsala University, Sweden.
    Teramura, Yuji
    Univ Tokyo, Japan;Uppsala University, Sweden.
    Modification of human MSC surface with oligopeptide-PEG-lipids for selective binding to activated endothelium2019In: Journal of Biomedical Materials Research. Part A, ISSN 1549-3296, E-ISSN 1552-4965, Vol. 107, no 8, p. 1779-1792Article in journal (Refereed)
    Abstract [en]

    Promising cell therapies using mesenchymal stem cells (MSCs) is proposed for stroke patients. Therefore, we aimed to efficiently accumulate human MSC (hMSC) to damaged brain area to improve the therapeutic effect using poly(ethylene glycol) (PEG)-conjugated phospholipid (PEG-lipid) carrying an oligopeptide as a ligand, specific for E-selectin which is upregulated on activated endothelial cells under hypoxia-like stroke. Here we synthesized E-selectin-binding oligopeptide (ES-bp) conjugated with PEG spacer having different molecular weights from 1 to 40 kDa. We found that ES-bp can be immobilized onto the hMSC surface through PEG-lipid without influence on cell growth and differentiation into adipocytes and osteocytes, respectively. It is also possible to control the immobilization of ES-bp on hMSC surface (<10(8) ES-bp per cell). Immobilized ES-bp can be continuously immobilized at the outside of cell membrane when PEG-lipids with PEG 5 and 40 kDa were used. In addition, the modified hMSC can specifically attach onto E-selectin-immobilized surface as a model surface of activated endothelium in human blood, indicating the sufficient number of immobilized ES-bp onto hMSC. Thus, this technique is one of the candidates for hMSC accumulation to cerebral infarction area. (c) 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1779-1792, 2019.

  • 172.
    Norrby, Marlene
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Gene and protein expression in denervated atrophic and hypertrophic skeletal muscle2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Following denervation skeletal muscles change their functional and structural properties. Some changes resemble conditions in developing muscles and may be important for reinnervation. Due to inactivity following denervation most skeletal muscles loose muscle mass and become atrophic. The hemidiaphragm muscle, however, undergoes a phase of transient hypertrophy following denervation, gaining weight during the first 6-10 days followed by a decrease in weight. In this thesis the expression (mRNA, protein and protein phosphorylations) of potential factors involved in the regulation of muscle mass were examined in denervated hind-limb and hemidiaphragm muscles.

    NIFK is a protein that associates with Ki67, a protein expressed predominantly in proliferating cells. The mRNA expression of NIFK was upregulated in denervated atrophic muscles but unaltered in denervated hypertrophic muscles, suggesting a potential role in the regulation of skeletal muscle mass (Paper I). p38 MAPK has previously been implicated in both anabolic and catabolic processes. Its substrate MK2 becomes phosphorylated at two sites, one of which is suggested to be important for nuclear export. MK2 phosphorylation at this site correlated with muscle weight in both atrophic and hypertrophic denervated muscles and may thus have a role in atrophy and hypertrophy (Paper III). Factors regulating protein synthesis are likely to play a role in atrophy and hypertrophy and many signaling pathways appear to converge on the formation of the translation initiation complex. The protein expression and phosphorylation status of several components in both Wnt and Akt signaling pathways indicate increased protein synthesis in denervated atrophic muscles as well as in denervated hypertrophic muscles (Papers II, IV and V). This suggests that increased protein degradation is more important than decreased protein synthesis for the loss of muscle mass in denervated atrophic muscles.

  • 173.
    Näsström, Thomas
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Andersson, Per Ola
    FOI Swedish Defence Research Agency, Sweden, Sweden;Uppsala University, Sweden.
    Lejon, Christian
    FOI Swedish Defence Research Agency, Sweden, Sweden.
    Karlsson, Björn C. G.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Amyloid fibrils prepared using an acetylated and methyl amidated peptide model of the alpha-Synuclein NAC 71-82 amino acid stretch contain an additional cross-beta structure also found in prion proteins2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, p. 1-14, article id 15949Article in journal (Refereed)
    Abstract [en]

    The 71-82 fragment of the non-amyloid-beta component (NAC) region of the Parkinson's disease (PD) and dementia with Lewy bodies (DLB) related protein alpha-Synuclein, has been reported to be important during protein misfolding. Although reports have demonstrated the importance of this fragment for the aggregation properties of the full-length protein, its exact role in pre-fibrillar oligomerisation, fibrillar growth and morphology has not yet been fully elucidated. Here, we provide evidence that fibrils prepared from an acetylated and methyl amidated peptide of the NAC 71-82 amino acid stretch of alpha-Synuclein are amyloid and contain, in addition to the cross-beta structure detected in the full-length protein fibrils, a cross-beta structure previously observed in prion proteins. These results shed light on the aggregation propensity of the NAC 71-82 amino acid stretch of the full-length protein but also the roles of the N- and C-terminal domains of alpha-Synuclein in balancing this aggregation propensity. The results also suggest that early aggregated forms of the capped NAC 71-82 peptide generated structures were stabilised by an anti-parallel and twisted beta-sheet motif. Due to its expected toxicity, this beta-sheet motif may be a promising molecular target for the development of therapeutic strategies for PD and DLB.

  • 174.
    Näsström, Thomas
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ådén, Jörgen
    Umeå University, Sweden.
    Shibata, Fumina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Andersson, Per Ola
    Uppsala University, Sweden.
    Karlsson, Björn C. G.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    A Capped Peptide of the Aggregation Prone NAC 71–82 Amino Acid Stretch of α-Synuclein Folds into Soluble β-Sheet Oligomers at Low and Elevated Peptide Concentrations2020In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 21, no 5, p. 1-14, article id 1629Article in journal (Refereed)
    Abstract [en]

    Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

  • 175. Ohlson, Sten
    Exploiting weak affinities1992In: Practical protein chromatography / [ed] Andrew Kenney and Susan Fowell, Totowa: Humana Press, 1992, 1, p. 197-208Chapter in book (Other academic)
    Abstract [en]

    Affinity chromatography (AC) (1) is a technique that is based on the specific interactions between two molecular species. The molecules involved are usually of biochemical nature, such as antibody-antigen, enzyme-inhibitor, and receptor-hormone.

  • 176. Ohlsson, Rolf
    et al.
    Brodelius, Peter
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Mosbach, Klaus
    Affinity Chromatography of Enzymes on an AMP-Analogue: Specific Elution of Dehydrogenases from a General Ligand1972In: FEBS letters, ISSN 0014-5793, Vol. 25, p. 234-238Article in journal (Refereed)
  • 177.
    Olofsson, Linda
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Engström, Alexander
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Lundgren, Anneli
    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.
    Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L.2011In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 11, article id 45Article in journal (Refereed)
    Abstract [en]

    BackgroundRecently, Artemisia annua L. (annual or sweet wormwood) has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues.

    ResultsThe four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13) reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures). Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves.

    ConclusionsFour genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on artemisinin production through reduction of dihydroartemisinic aldehyde to dihydroartemisinic alcohol. However, our results show that this enzyme is expressed only at low levels in tissues producing artemisinin and consequently its effect on artemisinin production may be limited. Finally, squalene synthase but not other sesquiterpene synthases appears to be a significant competitor for farnesyl diphosphate in artemisinin-producing tissues.

  • 178.
    Olofsson, Linda
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Lundgren, Anneli
    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.
    Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.2012In: Plant Science, ISSN 0168-9452, E-ISSN 1873-2259, Vol. 183, p. 9-13Article in journal (Refereed)
    Abstract [en]

    The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

  • 179.
    Olsson, Mikael E.
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Olofsson, Linda
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Lindahl, Ann-Louise
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Lundgren, Anneli
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Brodelius, Maria
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Brodelius, Peter E.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L2009In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Phytochemistry, Vol. 70, no 9, p. 1123-1128Article in journal (Refereed)
    Abstract [en]

    A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta 11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1 alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.

  • 180.
    Ooi, Saik Ann
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Investigation of toxin binding to bacterial voltage-gated sodium ion channels2018Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Voltage-gated sodium ion channels are membrane proteins that initiate the action potentials in nerves, endocrine and muscle cells. A group of selected neurotoxins and conotoxins were found to have an effect on the excitation and inhibition of the bacterial voltage-gated sodium channels. This fact provides an opportunity to obtain a more detailed description of the molecular mechanism by which the sodium channel governs the communication within cells, with the aid of the studied toxins. In order to investigate this possibility, we employed a combination of computational and experimental methods. Computational techniques aimed to provide the atomistic details of interaction between toxins and the sodium channel where the data obtained were correlated with experimental studies in vitro. Interestingly, among the studied toxins, the binding modes for neurotoxin (TX1) and conotoxin (TX5) were found to be different where TX1 revealed a higher binding affinity to the sodium channel in comparison with that observed for TX5 (docking and thermodynamic work calculations). An explanation to this behavior was believed to be due to the higher number of interaction points formed between TX1 and NavAb originating from different bound conformations of that toxin at the pore of the sodium channel. Initial experimental validation of TX1 binding to the sodium channel using surface plasmon resonance technique demonstrated concentration dependent binding. Based upon the observed behavior of TX1, an SPR binding assay can be developed. Altogether, the results obtained in this work not only reflect the complexity of toxin binding to sodium channels, but the potential of using a combination of computational methods and biochemical assay for the development of new protein drugs for therapy.

  • 181.
    Orozovic, Goran
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Resistance to neuraminidase inhibitors in influenza A virus isolated from mallards2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Influenza A virus belongs to the Orothomyxoviridae family of viruses and is one of the most common pathogens that cause infections of the respiratory tract. The aim of this thesis was to investigate if neuraminidase inhibitor (NAI) Tamiflu® (oseltamivir, OC) and Relenza® (zanamivir, ZA) - related resistance mutations exist in the neuraminidase (NA) gene of viruses collected from wild birds.

    A new set of degenerate primers was designed for the sequencing procedure, which resulted in a protocol that reduced time and costs of NA sequencing. This protocol was employed for subtyping of 120 NA genes (i.e. influenza viruses). Altogether, 230 NA sequences from avian influenza viruses originating from wild mallards (Ottenby, Sweden) were scanned for NAI-related mutations together with 5,490 avian, 379 swine and 122 environmental NA sequences from the NCBI dataset. The screening showed a distinction between the numbers of mutants found in avian virus sequences derived from NCBI (2.4%) as compared to virus sequences form mallards (6.5%). This is the first report of NAI resistance mutations in viruses isolated from wild birds.

    The mutants carrying NAI resistance-related and resistance-unrelated mutations were screened using NA inhibition assay (NAIA) with ZA and OC inhibitors. The majority of mutations assayed showed IC50 values indicating an inhibitor sensitive phenotype. One H12N3 mutant showed a cross-resistant phenotype, i.e. insensitive to both ZA and OC treatment. Protein structure homology-modeling indicated that this cross-resistance might be associated to a D151K mutation, possibly supported by changes in NA residue 149, 150, 152 and 153. In addition, an OC resistance-related emergence of H274Y mutants was revealed in an experimental set up where mallard ducks, subjected to different concentrations of OC ( 0.28, 3.5 and 280 nM)  in their water pool, were infected with avian H1N1 virus.

    In conclusion, this thesis provides new insights into the field of NAI resistance in avian influenza virus as well as indicating the evolutionary forces modern drug design has to confront. This thesis also emphasizes the importance of a continuous search for new means of protecting the human population from this potentially devastating pathogen. 

  • 182.
    Orozovic, Goran
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Latorre-Margalef, Neus
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Wahlgren, John
    Muradrasoli, Shaman
    Olsen, Björn
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene2010In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 170, no 1-2, p. 94-98Article in journal (Refereed)
    Abstract [en]

    This study describes the design of degenerate primers and their use for synthesis of full-length avian influenza A neuramindase (NA). Each reaction was performed using either two forward primers and one reverse primer, or one forward primer and one reverse primer. Both primer combinations had comparable amplification efficiencies for all NA subtypes (1-9). A total of 11 virus strains, including both field isolates and reference strains, were amplified successfully using these degenerate primer sets. Of the sequences amplified, 108 strains (93.9%) resulted in near full-length NA cDNAs after two readings with one forward primer and one reverse primer. Of the remaining sequences, five strains (4.3%) yielded reads with enough information for subtype categorization by BLAST although they were of insufficient quality for assembly. One strain (0.9%) yielded different subtypes from both sequence reads whereas the other one (0.9%) was not possible to assemble and subtype. This successful demonstration of these degenerate primers for the amplification and sequencing of all avian NA subtypes suggests that these primers could be employed in the avian influenza surveillance program as well as studies of antiviral resistance, virus ecology or viral phylogeny.

  • 183.
    Orrem, Hilde L.
    et al.
    Oslo Univ Hosp, Norway;Univ Oslo, Norway.
    Shetelig, Christian
    Univ Oslo, Norway;Oslo Univ Hosp Ulleval, Norway.
    Ueland, Thor
    Univ Oslo, Norway.;Oslo Univ Hosp, Norway;Univ Tromso, Norway.
    Limalanathan, Shanmuganathan
    Oslo Univ Hosp Ulleval, Norway;Feiring Heart Clin, Norway.
    Nilsson, Per H.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Univ Oslo, Norway.
    Husebye, Trygve
    Oslo Univ Hosp Ulleval, Norway;Univ Oslo, Norway.
    Aukrust, Pal
    Univ Oslo, Norway;Oslo Univ Hosp, Norway.
    Seljeflot, Ingebjorg
    Univ Oslo, Norway;Oslo Univ Hosp Ulleval, Norway.
    Hoffmann, Pavel
    Univ Oslo, Norway;Oslo Univ Hosp Ulleval, Norway.
    Eritsland, Jan
    Oslo Univ Hosp Ulleval, Norway.
    Mollnes, Tom E.
    Oslo Univ Hosp, Norway;Univ Tromso, Norway;Univ Oslo, Norway;Nordland Hosp, Norway;Norwegian Univ Sci, Norway.
    Andersen, Geir Oystein
    Oslo Univ Hosp Ulleval, Norway;Univ Oslo, Norway.
    Yndestad, Arne
    Univ Oslo, Norway;Oslo Univ Hosp, Norway.
    Soluble IL-1 receptor 2 is associated with left ventricular remodelling in patients with ST-elevation myocardial infarction2018In: International Journal of Cardiology, ISSN 0167-5273, E-ISSN 1874-1754, Vol. 268, p. 187-192Article in journal (Refereed)
    Abstract [en]

    Background: The inflammatory response following myocardial infarction (MI) is prerequisite for proper healing of infarcted tissue, but can also have detrimental effects on cardiac function. Interleukin (IL)-1 alpha and IL-1 beta are potent inflammatory mediators and their bioactivity is tightly regulated by IL-1 receptor antagonist (IL-1ra) and soluble (s) IL-1 receptors (R). We aimed to examine whether levels of soluble regulators of IL-1 signalling are changed during ST-elevation MI (STEMI) and their associations with parameters of cardiac injury and ventricular remodelling. Methods: Plasma levels of IL-1Ra, sIL-1R1, sIL-1R2 and sIL-1R accessory protein (sIL-1RAcP) were measured by immunoassays in repeated samples from patients with STEMI (n = 255) and compared to healthy controls (n=65). Results: IL-1Ra, sIL-1R1 and sIL-1R2 levels were all significantly elevated after STEMI, while levels of sIL-1RAcP were lower compared to controls. sIL-1R2 levels (at different time points) correlated positively with C-reactive protein, myocardial infarct size and change in indexed left ventricular end-diastolic and end-systolic volume (LVEDVi and LVESVi) measured by cardiac MR acutely and after 4 months, and negatively with LV ejection fraction. Patients with >median levels of sIL-1R2 in the acute phase were more likely to have increased change in LVEDVi and LVESVi. Importantly, sIL-1R2 remained significantly associated with change in LVEDVi and LVESVi also after adjustment for clinical covariates. Conclusion: Levels of sIL-1R2 are independently associated with parameters of LV adverse remodelling following STEMI. (C 18 Elsevier B.V. All rights reserved.

  • 184.
    Persson, Bengt L.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    The PHO regulon2004In: Encyclopedia of biological chemistry: Vol. 3 N-R / [ed] William J. Lennarz and M. Daniel Lane, Oxford: Academic Press, 2004, 1, , p. 262-265p. 262-265Chapter in book (Other academic)
  • 185.
    Persson, Bengt L.
    et al.
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm.
    Ahnström, G
    Rydström, J
    Energy-linked nicotinamide nucleotide transhydrogenase. Hydrodynamic properties and active form of purified and membrane-bound mitochondrial transhydrogenase from beef heart1987In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 259, no 2, p. 341-349Article in journal (Refereed)
    Abstract [en]

    The mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to minimal assembly of the purified enzyme and of the enzyme in the mitochondrial inner membrane. Studies of the hydrodynamic properties of the purified enzyme in the presence of 0.3% Triton X-100 allowed determination of the Stokes radius, sedimentation constant, partial specific volume, frictional ratio, and molecular weight. Under these conditions transhydrogenase existed as an inactive monomer, suggesting that monomerization may be accompanied by inactivation. Radiation inactivation was used to determine the functional molecular size of purified detergent-dispersed transhydrogenase and transhydrogenase in beef heart submitochondrial particles. Under these conditions the catalytic activity of both the purified and the membrane-bound enzyme was found to be catalyzed by a dimeric form of the enzyme. These results suggest for the first time that the minimal functional assembly of detergent-dispersed as well as membrane-bound transhydrogenase is a dimer, which is not functionally associated with, for example, complex I or ATPase. In addition, the results are consistent with the possibility that the two subunits of transhydrogenase are catalytically active in an alternating fashion according to a previously proposed half-of-the-sites reactivity model. 

  • 186.
    Persson, Bengt L.
    et al.
    Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Amsterdam The Netherlands.
    Berden, J
    Rydström, J
    van Dam, K
    ATP-driven transhydrogenase provides an example of delocalized chemiosmotic coupling in reconstituted vesicles and in submitochondrial particles1987In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 894, no 2, p. 239-251Article in journal (Refereed)
    Abstract [en]

    The mechanism of coupling between mitochondrial ATPase (EC 3.6.1.3) and nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) was studied in reconstituted liposomes containing both purified enzymes and compared with their behavior in submitochondrial particles. In order to investigate the mode of coupling between the transhydrogenase and the ATPase by the double-inhibitor and inhibitor-uncoupler methods, suitable inhibitors of transhydrogenase and ATPase were selected. Phenylarsine oxide and A3′-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-NAD+ were used as transhydrogenase inhibitors, whereas of the various ATPase inhibitors tested aurovertin was found to be the most convenient. The inhibition of the ATP-driven transhydrogenase activity was proportional to the inhibition of both the ATPase and the transhydrogenase. Inhibitor-uncoupler titrations showed an increased sensitivity of the coupled reaction towards carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) — an uncoupler that preferentially uncouples localized interactions, according to Herweijer et al. (Biochim. Biophys. Acta 849 (1986) 276–287) — when the primary pump was partially inhibited. However, when the secondary pump was partially inhibited the sensitivity towards FCCP remained unchanged. Similar results were obtained with submitochondrial particles. These results are in contrast to those obtained previously with the ATP-driven reverse electron flow. In addition, the amount of uncoupler required for uncoupling of the ATP-driven transhydrogenase was found to be similar to that required for the stimulation of the ATPase activity, both in reconstituted vesicles and in submitochondrial particles. Uncoupling of reversed electron flow to NAD+ required much less uncoupler. On the basis of these results, it is proposed that, in agreement with the chemiosmotic model, the interaction between ATPase and transhydrogenase in reconstituted vesicles as well as in submitochondrial particles occurs through the [...]. In contrast, the energy transfer between ATPase and NADH-ubiquinone oxidoreductase appears to occur via a more direct interaction, according to the above-mentioned results by Herweijer et al. 

  • 187.
    Persson, Bengt L.
    et al.
    the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm.
    Enander, K
    the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm.
    Tang, H-L
    Rydström, J
    the Department of Biochemistry, Arrhenius Laboratory, University of Stockholm.
    Energy-linked nicotinamide nucleotide transhydrogenase: Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography1984In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 259, p. 8626-8632Article in journal (Refereed)
    Abstract [en]

    Mitochondrial nicotinamide nucleotide transhydrogenasefrom beef heart was purified by a novel procedureinvolving fast protein liquid chromatography andcharacterized with respect to molecular and catalyticproperties. The method is reproducible, gives highlypure transhydrogenase as judged by silver staining,and can be modified to produce large amounts of puretranshydrogenase protein suitable for e.g. sequencingand other protein chemical studies.Transhydrogenase purified by fast protein liquidchromatography is reconstitutively active and pumpsprotons as indicated by an extensive quenching of 9-aminoacridine fluorescence. Under conditions whichgenerate a proton gradient in the absence of a membranepotential the activity of reconstituted transhydrogenaseis close to zero indicating a complete andproper incorporation in the membrane and a preferentialregulation of the enzyme by a proton gradientrather than a membrane potential.Treatment of reconstituted transhydrogenase withN,N-dicyclohexylcarbodiimide results in an inhibitionof proton pump activity without an effect on uncoupledcatalytic activity, suggestingt hat proton translocationand catalytic activities are not obligatory linked orthat this agent separates proton pumping from thecatalytic activity. 

  • 188.
    Persson, Bengt L.
    et al.
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm.
    Hartog, A F
    Rydström, J
    Berden, J
    NBD-Cl modification of essential residues in mitochondrial nicotinamide nucleotide transhydrogenase from beef heart1988In: Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, ISSN 0167-4838, E-ISSN 1879-2588, Vol. 953, p. 241-248Article in journal (Refereed)
    Abstract [en]

    Modification of mitochondrial nicotinamide nucleotide transhydrogenase (NADPH:NAD+ oxidoreductase, EC 1.6.1.1) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), followed by measurement of the absorption or fluorescence of the transhydrogenase-NBD adducts, resulted in a biphasic labelling of approx. 4–6 sulfhydryls, presumably cysteine residues. Of these 1–2 (27%) were fast-reacting and 3–4 (73%) slow-reacting sulfhydryls. In the presence of substrates, e.g., NADPH, the labelling was monophasic and all sulfhydryls were fast-reacting, suggesting that the modified sulfhydryls are predominantly localized peripheral to the NAD(P)(H)-binding sites. The rates of modification allowed the calculation of the rate constants for each phase of the labelling. Both in the absence and in the presence of a substrate, e.g., NADPH, the extent of labelling essentially parallelled the inhibition of transhydrogenase activity. Attempts to reactivate transhydrogenase by reduction of labelled sulfhydryls were not successful. Photo-induced transfer of the NBD adduct in partially inhibited transhydrogenase, from the sulfhydryls to reactive NH2 groups of amino-acid residue(s), identified as lysine residue(s), was parallelled by an inhibition of the residual transhydrogenase activity. It is suggested that a lysine localized close to the fast-reacting NBD-Cl-reactive sulfhydryl groups is essential for activity. 

  • 189.
    Persson, Bengt L.
    et al.
    UNIV STOCKHOLM, ARRHENIUS LAB, DEPT BIOCHEM.
    Rydström, J
    Evidence for a role of a vicinal dithiol in catalysis and proton pumping in mitochondrial nicotinamide nucleotide transhydrogenase1987In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 142, no 2, p. 573-578Article in journal (Refereed)
  • 190.
    Persson, Bengt L.
    et al.
    Department of Biochemistry and Biotechnology, Royal Institute of Technology .
    Rydström, J
    Kubista, M
    A circular dichroism study of mitochondrial transhydrogenase from beef heart1991In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 39, no 3, p. 267-272Article in journal (Refereed)
    Abstract [en]

    This paper describes a circular dichroism (CD) spectroscopy study of purified proton-pumping nicotinamide nucleotide transhydrogenase from beef heart. The CD spectrum obtained was used to estimate the content of secondary structures of the purified enzyme and suggests the presence of 40–45% α-helical structure and long, possibly membrane-spanning α-helices. The spectrum was essentially unaffected by the absence or presence of transhydrogenase substrates, suggesting that the catalytic and proton-translocating activities of the enzyme occur without major rearrangements at the level of secondary structures. 

  • 191.
    Persson, Bengt L.
    et al.
    UNIV STOCKHOLM, DEPT BIOCHEM.
    Teixera da Cruz, A
    Rydström, J
    Energy-dependent interaction of substrates with mitochondrial nicotinamide nucleotide transhydrogenase1986In: Chemica Scripta, ISSN 0004-2056, Vol. 26, no 4, p. 581-584Article in journal (Refereed)
  • 192.
    Persson, Malin
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Bengtsson, Elina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    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.
    Non-Linear Cross-Bridge Elasticity, ATP-Independent Detachment and ATP-Velocity Relationships for Skeletal Muscle Actomyosin2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 158A-158AArticle in journal (Other academic)
  • 193.
    Persson, Malin
    et al.
    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.
    Tolf, Conny
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Lindberg, A. Michael
    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.
    Kocer, Armagan
    University of Groningen, The Netherlands.
    Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, article id e55931Article in journal (Refereed)
    Abstract [en]

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (> 100 mu m) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  • 194. Petronilli, V
    et al.
    Persson, Bengt L.
    2Howard Hughes Medical Institute Research Laboratories, Molecular Biology Institute, University of California, Los Angeles, CA (U.S.A.).
    Zoratti, M
    Rydström, J
    Azzone, G F
    Flow-force relationships during energy transfer between mitochondrial proton pumps1991In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1058, no 2, p. 297-303Article in journal (Refereed)
    Abstract [en]

    The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, , and output flows of the ATPase, redox and transhydrogenase H+-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, , in an extended range of , independently of whether the H+-generating pump is the ATPase or a redox pump, or whether is depressed by inhibitors of the H+-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H+-pumps exhibit a steep dependence on . The flow-force relationships differ depending on whether the depression of is induced by inhibitors of the H+-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H+-consuming pump, at equivalent values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4−. With redox site I as proton-consuming pump, at equivalent values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4−. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H+-pumps. 

  • 195. Piatti, T
    et al.
    Boller, T
    Brodelius, Peter
    Institute of Biotechnology ETH-Hönggerberg CH-8093 Zürich, Switzerland .
    Induction of Ethylene Biosynthesis is Correlated with But Not Required for Induction of Alkaloids in Elicitor-treated Eschscholtzia Cells1991In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 30, no 7, p. 2151-2154Article in journal (Refereed)
    Abstract [en]

    Cell suspension cultures of Eschscholtzia californica produce almost no ethylene under normal growth conditions. Addition of 1-aminocyclopropane carboxylic acid (ACC) to the culture results in a strong stimu lation of ethylene formation, indicating that the ethylene-forming enzyme is constitutively present in the cells. Incubation of the cells with different amounts of an elicitor derived from yeast extract leads to a dose-dependent induction of ACC synthase and ethylene biosynthesis. This effect is correlated with the previously described induction of benzophenanthridine alkaloids. Aminoethoxyvinylglycine, a known inhibitor of ACC synthase, suppresses the induction of ethylene biosynthesis but does not affect induction of alkaloid accumulation, indicating that biosynthesis of ACC and ethylene are not required for induction of alkaloid accumulation. 

  • 196. Prenosil, J E
    et al.
    Hegglin, M
    Baumann, T W
    Frischknecht, P M
    Kappeler, A W
    Brodelius, Peter
    Swiss Federal Institute of Technology (ETH), Institute of Biotechnology, Honggerberg, CH-8093 Zurich, Switzerland.
    Haldimann, D
    Purine Alkaloid Producing Cell Cultures: Fundamental Aspects and Possible Applications in Biotechnology1987In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 9, no 8, p. 450-458Article in journal (Refereed)
    Abstract [en]

    Coffea arabica is one of the plant species that has been widely studied with attention largely being given to its secondary products, caffeine and other purine alkaloids. The biosynthesis and significance of these alkaloids for the plant are elucidated and presented. Tissue cell culture and fundamental aspects of cell growth and alkaloid productivity are also discussed. The feasibility of Coffea cultivation in cell suspension has recently attracted the interest of many researchers. Although this cultivation is not of commercial interest, Coffea is especially suitable as a model cell line for reaction engineering studies because the purine alkaloids are well-characterised and readily released in culture medium. The use of free and immobilized coffee cells in various types of bioreactors (stirred tank, expanded bed, and membrane device) is shown. 

  • 197.
    Rahman, Mohammad A.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rassier, Dilson
    McGill Univ, Canada.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Dissecting Actomyosin Mechanochemistry using Blebbistatin as a Pharmacological Tool2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 117A-117AArticle in journal (Other academic)
  • 198.
    Rahman, Mohammad A.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Lund University, Sweden.
    Reuther, Cordula
    Tech Univ Dresden, Germany;Max Planck Inst Mol Cell Biol & Genet, Germany.
    Lindberg, Frida W.
    Lund University, Sweden.
    Mengoni, Martina
    Tech Univ Dresden, Germany;Max Planck Inst Mol Cell Biol & Genet, Germany.
    Salhotra, Aseem
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Lund University, Sweden.
    Heldt, Georg
    Fraunhofer Inst Elect Nano Syst, Germany.
    Linke, Heiner
    Lund University, Sweden.
    Diez, Stefan
    Tech Univ Dresden, Germany;Max Planck Inst Mol Cell Biol & Genet, Germany.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Lund University, Sweden.
    Regeneration of Assembled, Molecular-Motor-Based Bionanodevices2019In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 19, no 10, p. 7155-7163Article in journal (Refereed)
    Abstract [en]

    The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.

  • 199. Ramos-Soriano, Javier
    et al.
    Niss, Ulf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Angulo, Jesus
    Angulo, Manuel
    Moreno-Vargas, Antonio J.
    Carmona, Ana T.
    Ohlson, Sten
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Robina, Inmaculada
    Synthesis, Biological Evaluation, WAC and NMR Studies of S-Galactosides and Non-Carbohydrate Ligands of Cholera Toxin Based on Polyhydroxyalkylfuroate Moieties2013In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 19, no 52, p. 17989-18003Article in journal (Refereed)
    Abstract [en]

    The synthesis of several non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties is reported. Some of them have been linked to D-galactose through a stable and well-tolerated S-glycosidic bond. They represent a novel type of non-hydrolyzable bidentate ligand featuring galactose and polyhydroxyalkylfuroic esters as pharmacophoric residues, thus mimicking the GM1 ganglioside. The affinity of the new compounds towards cholera toxin was measured by weak affinity chromatography (WAC). The interaction of the best candidates with this toxin was also studied by saturation transfer difference NMR experiments, which allowed identification of the binding epitopes of the ligands interacting with the protein. Interestingly, the highest affinity was shown by non-carbohydrate mimics based on a polyhydroxyalkylfuroic ester structure.

  • 200.
    Refaat, Doaa
    et al.
    Agr Res Ctr, Egypt;Beni Suef Univ, Egypt.
    Aggour, Mohamed G.
    Agr Res Ctr, Egypt.
    Farghali, Ahmed A.
    Beni Suef Univ, Egypt.
    Mahajan, Rashmi
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Wiklander, Jesper G.
    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.
    Piletsky, Sergey A.
    Univ Leicester, UK.
    Strategies for Molecular Imprinting and the Evolution of MIP Nanoparticles as Plastic Antibodies-Synthesis and Applications2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 24, p. 1-21, article id 6304Article, review/survey (Refereed)
    Abstract [en]

    Materials that can mimic the molecular recognition-based functions found in biology are a significant goal for science and technology. Molecular imprinting is a technology that addresses this challenge by providing polymeric materials with antibody-like recognition characteristics. Recently, significant progress has been achieved in solving many of the practical problems traditionally associated with molecularly imprinted polymers (MIPs), such as difficulties with imprinting of proteins, poor compatibility with aqueous environments, template leakage, and the presence of heterogeneous populations of binding sites in the polymers that contribute to high levels of non-specific binding. This success is closely related to the technology-driven shift in MIP research from traditional bulk polymer formats into the nanomaterial domain. The aim of this article is to throw light on recent developments in this field and to present a critical discussion of the current state of molecular imprinting and its potential in real world applications.

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