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  • 1.
    Andersson, Håkan S.
    University of Kalmar, School of Pure and Applied Natural Sciences. Lund University, Sweden.
    On the Nature and Origin of Recognition in Molecularly Imprinted Polymers.: Paths Towards Their Rational Design.1997Licentiate thesis, monograph (Other academic)
  • 2.
    Andersson, Håkan S.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Towards the Rational Design of Molecularly Imprinted Polymers1999Doctoral thesis, comprehensive summary (Other academic)
  • 3.
    Andersson, Håkan S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Jacobsson, Erik
    Uppsala University.
    Eriksson, Camilla
    Uppsala University.
    Hedström, Martin
    Lund University.
    Seth, Henrik
    University of Gothenburg.
    Sundberg, Per
    University of Gothenburg.
    Rosengren, Johan
    University of Queensland.
    Strand, Malin
    Swedish University of Agricultural Sciences.
    Göransson, Ulf
    Uppsala University.
    The toxicity of ribbon worms: alpha-nemertides or tetrodotoxin, or both?2016In: Planta Medica, ISSN 0032-0943, E-ISSN 1439-0221, Vol. 82, no Supplement 1, P549Article in journal (Other academic)
    Abstract [en]

    The marine ribbon worms (nemerteans) are predators which capture their prey by everting a proboscis carrying a mixture of toxins which brings on rapid paralysis [1]. Moreover, ribbon worms have a thick layer of epidermal mucus of similar constitution. Tetrodotoxin (TTX) has been identified as one of these toxins [2]. The extreme toxicity of TTX (lethal by ingestion of 0.5-2 mg) is due to its ability to block voltage-gated sodium channels. Although several bacterial species (among these Vibrio sp.) have been linked to its synthesis, the biogenic origin and biosynthesis is unclear. One hypothesis is that TTX production occurs in a symbiotic relationship with its host, in this case the ribbon worm [3]. We have made significant effort to identify TTX in a setup for production through the cultivation of Vibrio alginolyticus in nutrient broth infused with mucus from the ribbon worm Lineus longissimus. Toxicity was demonstrated by fraction injections into shore crabs, but no TTX was found, and it could be shown conclusively that toxicity was unrelated to TTX and the Vibrio culture itself, and rather a constituent of the ribbon worm mucus [4]. The following studies led us to the discovery of a new class of peptides, the alpha-nemertides, in the mucus of the ribbon worms, which could be directly linked to the toxic effects. A literature review of the available evidence for TTX in ribbon worms show that the evidence in most cases are indirect, although notable exceptions exist. This points to the necessity to further investigate the presence and roles of TTX and alpha-nemertides in ribbon worms.

  • 4.
    Andersson, Håkan S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Jacobsson, Erik
    Uppsala University.
    Rosengren, Johan
    University of Queensland, Australia.
    Strand, Malin
    Swedish University of Agricultural Sciences.
    Göransson, Ulf
    Uppsala University.
    Discovery of novel ion-channel active peptide toxins in a North Sea Ribbon Worm2016Conference paper (Other academic)
    Abstract [en]

    Ribbon worms (nemerteans) are marine predators, which capture their prey using a proboscis containing a mixture of toxins which brings on rapid paralysis [1]. In addition, their epidermis contains thick mucus of similar toxic constitution. One very potent toxin reported in ribbon worm mucus is tetrodotoxin (TTX). However, despite significant efforts, Strand et al. [2] were unable to detect any TTX, neither in the mucus of the ribbon worm Lineus longissimus, nor from Vibrio alginolyticus cultures isolated from and cultivated in the mucus. These observations challenged the notion of general presence of TTX in ribbon worm mucus, and prompted us to look for other toxins [3]. Using LC-MS analysis of mucus extracts, we identified three peptides present in significant amounts. The peptides were sequenced using a combination of MS/MS analysis and transcriptomics, and whereas one of them strongly resembles the only peptide toxin previously characterized from ribbon worms, Neurotoxin B-IV [4], the other two were found to represent a previously unknown class of peptide toxins. The most abundant of these was synthesized, and its 3D structure determined. Preliminary toxicity tests on shore crab (C. maenas) indicated toxicity (through paralysis) on par with that of TTX. Further analyses have indicated that its toxic effects are due to binding to voltage sensitive sodium channels.

     

    With L. longissimus as our primary target, we are now mapping the presence of peptide toxins in ribbon worms, with the objectives to establish routes for synthesis, and to characterize the biological activities and structures of these peptides. The number of peptides of this novel class is increasing, and synthesis and characterization is well underway. The striking potencies of these peptides make them potentially amenable as novel insecticidal or anthelmintic leads, pharmacological tools or in biotechnology applications.

     

    References

    1. Strand M, Sundberg P. Nationalnyckeln till Sveriges flora och fauna [DO-DP]. Stjärnmaskar-Slemmaskar: Sipuncula-Nemertea: Artdatabanken, SLU; 2010.

    2. Strand M, Hedstrom M, Seth H, McEvoy EG, Jacobsson E, Goransson U, Andersson HS, Sundberg P. The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus-Just a False Positive? Marine Drugs. 2016;14(4).

    3. Strand M, Andersson HS. Slemmaskens hemlighet. Forskning & Framsteg. 2016;(2):26-33.

    4. Blumenthal KM, Kem WR. Structure and action of heteronemertine polypeptide toxins. Primary structure of Cerebratulus lacteus toxin B-IV. The Journal of Biological Chemistry. 1976;251(19):6025-9.

  • 5.
    Andersson, Johan
    Växjö University, Faculty of Mathematics/Science/Technology, School of Technology and Design.
    Hantering av bränsleflis: Enkätundersökning och bedömning av kvalitetsaspekter2008Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
    Abstract [sv]

    Med globalt ökat klimathot och ett ökat användande av biologiskt nedbrytbara material för att bland annat driva vår motorpark och värma våra hus kommer även ett krav på ökat produktion av utsläppsreducerande (koldioxid CO2) material så som biobränsle.

    För att kunna reducera utsläppen krävs inte bara att vi ökar produktionen av biologiskt nedbrytbart ämnen utan att dessa material har de egenskaperna som de förbränningssystem de används till optimalt kräver.

    Ett av de biologiskt nedbrytbara materialen är biobränsle, framställt av skogsavfall (grot). Dess hantering ifrån avverkningstillfället tills in- transport till förbränningen har varit kärnan i detta arbete. De två grundfrågorna som har varit själva motorn i arbetet har kretsat kring;

    o Vem äger vad i olika hanteringssteg?

    o Vem vet vad i olika hanteringssteg?

    Syftet med denna undersökning var att bland annat försöka få reda på svaren på frågorna ovan men även att väcka ett ökat intresse och förståelse kring hanteringen av biobränslet från avverkningstillfället fram till in- transport till en terminal alternativt direkt in till panncentralen.

    Undersökningen gjordes genom en enkätundersökning på Internet. Enkäten skickades ut till berörda parter i Sverige per e-post och adressaterna kunde svara direkt i datorn och skicka tillbaka enkäten utan att skriva ut den. Svaren från Skåne i söder upp till Mälardalen i norr vilket gör att man kan säga att denna undersökning gäller för södra Sverige.

  • 6.
    Andersson, Michael R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Functional aspects of inorganic phosphate transport2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Inorganic phosphate is an essential nutrient for all organisms. It is required for many cellular components as nucleic acids and phospholipids, and as energy-carrying compounds such as ATP. Thus, a regulated uptake of this pivotal nutrient is of outermost importance. Depending of the availability of phosphate in the surroundings the yeast Saccharomyces cerevisiae make use of two different systems for transporting phosphate into the interior of the cell: a low-affinity system that is active during surplus phosphate conditions and a high-affinity system that is active when the availability becomes limited. This thesis focuses on the high-affinity system, which is comprised of the Pho84 and Pho89 transporters. Of the two transporters, Pho84 is the predominant one, responsible for almost all phosphate uptake during low phosphate conditions, and the contribution of Pho89 is of minor importance. Hence Pho84 is by far the most well characterized phosphate transporter. Even though much is known about phosphate transporters in yeast little in known about how phosphate is transported. The work in this thesis aims to broaden the knowledge about the transport mechanism by the means of site-directed mutagenesis and functional characterization. Also the similarity of Pho84 to glucose sensors and the potential role of conserved residues in phosphate signaling are investigated. By the use of a high-affinity system deletion strain (∆Pho84 ∆Pho89), we also managed to investigate the functional importance of well conserved residues in Pho89. In summary: the work presented in this thesis has contributed to increase the knowledge about transport mechanisms in phosphate transporters.

  • 7.
    Andersson, Michael R.
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Samyn, Dieter R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Mutational analysis of conserved glutamic acids of Pho89, a Saccharomyces cerevisiae high-affinity inorganic phosphate:Na+ symporter2012In: Biologia (Bratislava), ISSN 0006-3088, E-ISSN 1336-9563, Vol. 67, no 6, 1056-1061 p.Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, the high-affinity phosphate transport system comprises the Pho84 and Pho89 permeases. The Pho89 permease catalyzes import of inorganic phosphate in a symport manner by utilizing Na+ ions as co-solute. We have addressed the functional importance of two glutamic acid residues at positions 55 and 491. Both residues are highly conserved amongst members of the inorganic phosphate transporter (PiT) family, which might be an indication of functional importance. Moreover, both residues have been shown to be of critical importance in the hPit2 transporter. We have created site-directed mutations of both E55 and E491 to lysine and glutamine. We observed that in all four cases there is a dramatic impact on the transport activity, and thus it seems that they indeed are of functional importance. Following these observations, we addressed the membrane topology of this protein by using several prediction programs. TOPCONS predicts a 7-5 transmembrane segment organization, which is the most concise topology as compared to the hPiT2 transporter. By understanding the functionality of these residues, we are able to correlate the Pho89 topology to that of the hPiT2, and can now further analyze residues which might play a role in the transport activity.

  • 8.
    Asif, Sana
    et al.
    Uppsala University.
    Jonsson, Nina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    Teramura, Yuji
    Univ Tokyo, Japan.
    Gustafson, Elisabet
    Univ Uppsala Hosp.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala University.
    Nilsson, Bo
    Uppsala University.
    Conjugation of human recombinant CD39 to primary human hepatocytes protects against thromboinflammation2015In: Xenotransplantation, ISSN 0908-665X, E-ISSN 1399-3089, Vol. 22, S87-S87 p.Article in journal (Other academic)
  • 9. Barry, S
    et al.
    Brodelius, Peter
    UNIV LUND, CTR CHEM, DEPT PURE & APPL BIOCHEM, S-22007 LUND 7, SWEDEN .
    Mosbach, K
    General Ligand Affinity Chromatography: N6-(6-Aminohexyl) 3',5'-ADP Sepharose as an Affinity Adsorbent for the CoA-Dependent Enzyme, Succinate Thiokinase1976In: FEBS letters, Vol. 70, no 1, 261-266 p.Article in journal (Refereed)
  • 10.
    Bengtsson, Elina
    et al.
    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.
    Kumar, Saroj
    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.
    Altered Structural State of Actin Filaments Upon MYOSIN II Binding2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2 Supplement 1, 299A-300A p., 1499-PosArticle in journal (Other academic)
  • 11.
    Bengtsson, Elina
    et al.
    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.
    Månsson, Alf
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Analysis of Flexural Rigidity of Actin Filaments Propelled by Surface Adsorbed Myosin Motors2013In: Cytoskeleton, ISSN 1949-3584, Vol. 70, no 11, 718-728 p.Article in journal (Refereed)
    Abstract [en]

    Actin filaments are central components of the cytoskeleton and the contractile machinery of muscle. The filaments are known to exist in a range of conformational states presumably with different flexural rigidity and thereby different persistence lengths. Our results analyze the approaches proposed previously to measure the persistence length from the statistics of the winding paths of actin filaments that are propelled by surface-adsorbed myosin motor fragments in the in vitro motility assay. Our results suggest that the persistence length of heavy meromyosin propelled actin filaments can be estimated with high accuracy and reproducibility using this approach provided that: (1) the in vitro motility assay experiments are designed to prevent bias in filament sliding directions, (2) at least 200 independent filament paths are studied, (3) the ratio between the sliding distance between measurements and the camera pixel-size is between 4 and 12, (4) the sliding distances between measurements is less than 50% of the expected persistence length, and (5) an appropriate cut-off value is chosen to exclude abrupt large angular changes in sliding direction that are complications, e.g., due to the presence of rigor heads. If the above precautions are taken the described method should be a useful routine part of in vitro motility assays thus expanding the amount of information to be gained from these. (c) 2013 Wiley Periodicals, Inc.

  • 12.
    Bengtsson, Elina
    et al.
    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. Karolinska Institutet ; McGill Univ, Canada.
    Rahman, Mohammad A.
    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 Technol Univ, India.
    Takatsuki, Hideyo
    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.
    Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 7, 1465-1477 p.Article in journal (Refereed)
    Abstract [en]

    Actin filaments have key roles in cell motility but are generally claimed to be passive interaction partners in actin-myosin -based motion generation. Here, we present evidence against this static view based on an altered myosin-induced actin filament gliding pattern in an in vitro motility assay at varied [MgATP]. The statistics that characterize the degree of meandering of the actin filament paths suggest that for [MgATP] >= 0.25 mM, the flexural rigidity of heavy meromyosin (HMM)-propelled actin filaments is similar (without phalloidin) or slightly lower (with phalloidin) than that of HMM-free filaments observed in solution without surface tethering. When [MgATP] was reduced to <= 0.1 mM, the actin filament paths in the in vitro motility assay became appreciably more winding in both the presence and absence of phalloidin. This effect of lowered [MgATP] was qualitatively different from that seen when HMM was mixed with ATP-insensitive, N-ethylmaleimide-treated HMM (NEM-HMM; 25-30%). In particular, the addition of NEM-HMM increased a non-Gaussian tail in the path curvature distribution as well as the number of events in which different parts of an actin filament followed different paths. These effects were the opposite of those observed with reduced [MgATP]. Theoretical modeling suggests a 30-40% lowered flexural rigidity of the actin filaments at [MgATP] <= 0.1 mM and local bending of the filament front upon each myosin head attachment. Overall, the results fit with appreciable structural changes in the actin filament during actomyosin-based motion generation, and modulation of the actin filament mechanical properties by the dominating chemomechanical actomyosin state.

  • 13.
    Bergman, I. M.
    et al.
    Aarhus Univ.
    Edman, Kjell
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    van As, P.
    Res & Technol Ctr, Technol & Serv BV, Hendrix Genet Res, Boxmeer, Netherlands.
    Huisman, A.
    Res & Technol Ctr, Technol & Serv BV, Hendrix Genet Res, Boxmeer, Netherlands.
    Juul-Madsen, Helle Risdahl
    Aarhus Univ.
    A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs2014In: Immunogenetics, ISSN 0093-7711, E-ISSN 1432-1211, Vol. 66, no 3, 171-184 p.Article in journal (Refereed)
    Abstract [en]

    The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2) affect mRNA expression, serum concentration, and susceptibility to disease, but the combinatory effect of pMBL1 and pMBL2 genotypes needs further elucidation. In the present study, pMBL1 and pMBL2 alleles, combined pMBL haplotypes, and MBL-A concentration in serum were analyzed in purebred Landrace (N = 30) and Duroc (N = 10) pigs. Furthermore, the combined pMBL haplotypes of 89 PiStrain x (Large White x Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1-previously shown to have profound effect on MBL-A concentration even in the heterozygote condition-was detected in 47 % of the animals. Finally, an association was found between low-producing MBL genotypes and low body weight on the day of weaning in the same animals.

  • 14.
    Bergström, Maria
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Liu, Shuang
    Kiick, Kristi
    Ohlson, Sten
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Cholera toxin inhibitors studied with High-performance liquid affinity chromatography: arobust method to evaluate receptor-ligand interactions2009In: Chemical Biology and Drug Design, ISSN 1747-0277, E-ISSN 1747-0285, Vol. 73, no 1, 132-141 p.Article in journal (Refereed)
    Abstract [en]

    Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The K(D) values of galactose and meta-nitrophenyl alpha-d-galactoside were determined with weak affinity chromatography to be 52 and 1 mm, respectively, which agree well with IC(50) values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but K(D) values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct K(D) values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis.

  • 15.
    Bjelic, Sinisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Protein carriers for passage of the Blood-Brain Barrier2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, 177-177 p.Article in journal (Other academic)
  • 16.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Aqvist, Johan
    Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 46, 14521-14528 p.Article in journal (Refereed)
    Abstract [en]

    The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment.

  • 17.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Brandsdal, Bjørn O
    University of Tromsø, Norway.
    Åqvist, Johan
    Uppsala University.
    Cold adaptation of enzyme reaction rates2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 38, 10049-10057 p.Article in journal (Refereed)
    Abstract [en]

    A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.

  • 18.
    Bjelic, Sinisa
    et al.
    University of Washington, USA.
    Kipnis, Yakov
    University of Washington, USA.
    Wang, Ling
    University of Washington, USA.
    Pianowski, Zbigniew
    ETH Zurich, Switzerland.
    Vorobiev, Sergey
    Columbia University, USA.
    Su, Min
    Columbia University, USA.
    Seetharaman, Jayaraman
    Columbia University, USA.
    Xiao, Rong
    The State University of New Jersey, USA.
    Kornhaber, Gregory
    The State University of New Jersey, USA ; University of Medicine and Dentistry of New Jersey, USA ; Northeast Structural Genomics Consortium, USA.
    Hunt, John F
    Columbia University, USA.
    Tong, Liang
    Columbia University, USA.
    Hilvert, Donald
    ETH Zurich, Switzerland.
    Baker, David
    University of Washington, USA.
    Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design2014In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 1, 256-271 p.Article in journal (Refereed)
    Abstract [en]

    Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

  • 19.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Nervall, M
    Uppsala University.
    Gutiérrez-de-Terán, H
    Uppsala University.
    Ersmark, K
    Uppsala University.
    Hallberg, A
    Uppsala University.
    Aqvist, J
    Uppsala University.
    Computational inhibitor design against malaria plasmepsins2007In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 64, no 17, 2285-2305 p.Article in journal (Refereed)
    Abstract [en]

    Plasmepsins are aspartic proteases involved in the degradation of the host cell hemoglobin that is used as a food source by the malaria parasite. Plasmepsins are highly promising as drug targets, especially when combined with the inhibition of falcipains that are also involved in hemoglobin catabolism. In this review, we discuss the mechanism of plasmepsins I-IV in view of the interest in transition state mimetics as potential compounds for lead development. Inhibitor development against plasmepsin II as well as relevant crystal structures are summarized in order to give an overview of the field. Application of computational techniques, especially binding affinity prediction by the linear interaction energy method, in the development of malarial plasmepsin inhibitors has been highly successful and is discussed in detail. Homology modeling and molecular docking have been useful in the current inhibitor design project, and the combination of such methods with binding free energy calculations is analyzed.

  • 20.
    Bjelic, Sinisa
    et al.
    University of Washington, USA.
    Nivón, Lucas G
    University of Washington, USA.
    Çelebi-Ölçüm, Nihan
    University of California, USA ; Yeditepe University, Turkey.
    Kiss, Gert
    University of California, USA.
    Rosewall, Carolyn F
    Lovick, Helena M
    Ingalls, Erica L
    Gallaher, Jasmine Lynn
    University of Washington, USA.
    Seetharaman, Jayaraman
    Columbia University, USA.
    Lew, Scott
    Columbia University, USA.
    Montelione, Gaetano Thomas
    Columbia University, USA.
    Hunt, John Francis
    Columbia University, USA.
    Michael, Forrest Edwin
    Houk, K N
    University of California, USA.
    Baker, David
    University of Washington, USA.
    Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction2013In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 8, no 4, 749-757 p.Article in journal (Refereed)
    Abstract [en]

    The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.

  • 21.
    Bjelic, Sinisa
    et al.
    Uppsala University.
    Åqvist, Johan
    Uppsala University.
    Catalysis and linear free energy relationships in aspartic proteases2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 25, 7709-7723 p.Article in journal (Refereed)
    Abstract [en]

    Aspartic proteases are receiving considerable attention as potential drug targets in several serious diseases, such as AIDS, malaria, and Alzheimer's disease. These enzymes cleave polypeptide chains, often between specific amino acid residues, but despite the common reaction mechanism, they exhibit large structural differences. Here, the catalytic mechanism of aspartic proteases plasmepsin II, cathepsin D, and HIV-1 protease is examined by computer simulations utilizing the empirical valence bond approach in combination with molecular dynamics and free energy perturbation calculations. Free energy profiles are established for four different substrates, each six amino acids long and containing hydrophobic side chains in the P1 and P1' positions. Our simulations reproduce the catalytic effect of these enzymes, which accelerate the reaction rate by a factor of approximately 10(10) compared to that of the corresponding uncatalyzed reaction in water. The calculations elucidate the origin of the catalytic effect and allow a rationalization of the fact that, despite large structural differences between plasmepsin II/cathepsin D and HIV-1 protease, the magnitude of their rate enhancement is very similar. Amino acid residues surrounding the active site together with structurally conserved water molecules are found to play an important role in catalysis, mainly through dipolar (electrostatic) stabilization. A linear free energy relationship for the reactions in the different enzymes is established that also demonstrates the reduced reorganization energy in the enzymes compared to that in the uncatalyzed water reaction.

  • 22. Bramble, J L
    et al.
    Graves, D J
    Brodelius, Peter
    Department of Plant Biotechnology, University of Lund.
    Calcium and Phosphate Effects on Growth and Alkaloid Production in Coffea arabica: Experimental Results and Mathematical Model.1991In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 37, no 9, 859-868 p.Article in journal (Refereed)
    Abstract [en]

    Plant, mammalian, and microbial cells are commonly immobilized in calcium alginate gels for the production of valuable secondary metabolites. However, calcium ions are known to inhibit growth in various type of cells, and calcium is an integral part of such gels. Therefore, an investigation was conducted to evaluate the effect of calcium on the growth and alkaloid production of a model cell-line, Coffea arabica, in suspension culture before attempting to immobilize such cells in alginate. A kinetic model was then developed from the results to describe cell growth and alkaloid production and the mechanism by which calcium influences these variables. In addition, it was observed that there was a characteristic relationship between the concentration of calcium in the external medium and the concentration of extracellular and intracellular phosphate. The intracellular phosphate level was, in turn, related to the production of alkaloids. Using these results, a dynamic mathematical model of cell growth and alkaloid production was developed based on the proposed roles of calcium and phosphate. The model showed satisfactory agreement with three sets of experiments at different calcium concentrations. A possible linkage between the calcium and phosphate results is postulated based on the limited solubility of calcium phosphate. 

  • 23. Bramble, J L
    et al.
    Graves, D J
    Brodelius, Peter
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Plant Cell Culture using a Novel Bioreactor: The Magnetically Stabilized Fluidized Bed1990In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 6, no 6, 452-457 p.Article in journal (Refereed)
    Abstract [en]

    A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture. 

  • 24.
    Brodelius, Peter
    Department of Plant Biochemistry, University of Lund .
    Enzyme assays1991In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 2, no 1, 23-29 p.Article in journal (Refereed)
    Abstract [en]

    The past year or so has seen the development of new enzyme assays, as well as the improvement of existing ones. Assays are becoming more rapid and sensitive as a result of modifications such as amplification of the enzyme product(s). Recombinant DNA technology is now being recognized as a particularly useful tool in the search for improved assay systems. 

  • 25.
    Brodelius, Peter
    University of Kalmar, School of Pure and Applied Natural Sciences.
    High-performance Liquid Chromatographic Analysis of Analogous Amino and Oxo Acids for the Determination of Amino Acid Oxidase and Transaminase Activities1984In: Acta chemica Scandinavica, Series B - Organic Chemistry and Biochemistry, Vol. 38, no 3, 219-223 p.Article in journal (Refereed)
  • 26.
    Brodelius, Peter
    Institute of Biotechnology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Ziirich, Switzerland.
    Permeabilization of Plant Cells for Release of Intracellularly Stored Products: Viability Studies1988In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 27, 561-566 p.Article in journal (Refereed)
    Abstract [en]

    The effects of various chemical substanceson the permeability of plasma membranesand tonoplasts of three suspension cultures (Catharanthusroseus, Thalictrum rugosum and Chenopodiumrubrum) have been studied. The permeabilityof the plasma membrane is monitoredby measuring the activity of the cytosolic enzymeisocitrate dehydrogenase and the permeability ofthe tonoplast is measured by determining the releaseof substances stored in the vacuoles (inorganicphosphate, berberine and betanin for thethree cell lines, respectively). The minimum concentrationrequired for quantitative release of vacuolarproducts have been established for five differentpermeabilization agents. Cell viability islost upon permeabilization except for treatmentof Catharanthus roseus with DMSO and Triton X-100. 

  • 27.
    Brodelius, Peter
    Institute of Biotechnology, Swiss Federal Institute of Technology, Hönggerberg, CH-8093 Zürich, Switzerland.
    The Potential Role of Immobilization in Plant Cell Biotechnology1985In: Trends in Biotechnology, ISSN 0167-7799, E-ISSN 1879-3096, Vol. 3, no 11, 280-285 p.Article in journal (Refereed)
    Abstract [en]

    It has long been hoped that the unique biosynthetic capacity of plants could be exploited in vitro using culture systems analogous to microbial fermentations. However, the characteristics of both the growth and metabolism of plant cells in vitro differ considerably from those of microbial cells and plant cell suspension culture systems have met with limited success. Immobilizing the cells creates a new set of options for the plant biotechnologist to explore. Improvements in some process criteria are apparent although evaluating the potential of immobilized plant cells for producing commercial compounds will only be possible when the biological problems have been overcome. 

  • 28.
    Brodelius, Peter
    et al.
    Biochemistry 2, University of Lund, Chemical Center, P. O. B. 740, S-22007 Lund 7, Sweden.
    Deus, B
    Mosbach, K
    Zenk, M H
    Immobilized Plant Cells for the Production and Transformation of Natural Products1979In: FEBS letters, Vol. 103, no 1, 93-97 p.Article in journal (Refereed)
  • 29.
    Brodelius, Peter
    et al.
    Institute of Biotechnology ETH-Hönggerberg CH-8093 Zürich, Switzerland.
    Funk, C
    Häner, A
    Villegas, Mirza
    A Procedure for the Determination of Optimal Chitosan Concentrations for Elicitation of Cultured Plant Cells.1989In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 28, no 10, 2651-2654 p.Article in journal (Refereed)
    Abstract [en]

    An experimental method for determination of the optimum chitosan concentration for elicitation of plant cell suspension cultures is presented. The procedure, which is based on measurements of the conductivity of the culture medium after addition of chitosan, has been applied to suspension cultures of Nicotiana tabacum and Eschscholtzia californica. Increased conductivity of the medium (due to permeabilization of the cells) results in decreased secondary product formation and cell growth. Maximum product formation is observed for cells elicited with the highest chitosan concentration which does not affect membrane permeability. 

  • 30.
    Brodelius, Peter
    et al.
    Institute of Biotechnology, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Ziirich, Switzerland.
    Funk, C
    Shillito, R D
    Permeabilization of Cultivated Plant Cells by Electroporation for Release of Intracellularly Stored Secondary Products1988In: Plant Cell Reports, ISSN 0721-7714, E-ISSN 1432-203X, Vol. 7, no 3, 186-188 p.Article in journal (Refereed)
    Abstract [en]

    Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells. 

  • 31.
    Brodelius, Peter
    et al.
    Department of Chemistry, Q-058, University of California at San Diego, La Jolla, California 92093, U.S.A.
    Kaplan, N O
    Studies of Bovine Liver Glutamate Dehydrogenase by Analytical Affinity Chromatography on Immobilized AMP Analogs1979In: Archives of Biochemistry and Biophysics, Vol. 194, no 2, 449-456 p.Article in journal (Refereed)
    Abstract [en]

    Bovine liver glutamate dehydrogenase has been studied by analytical affinity chromatography on two immobilized AMP analogs, i.e., N6-(6-aminohexyl)-AMP and 8-(6-aminohexyl)-amino-AMP. The existence of various enzyme-coenzyme and enzyme-effector complexes has been verified. Also the cooperative formation of two ternary complexes, i.e., glutamic dehydrogenase (GHD)-NADP-glutamate and GDH-ADP-leucine, has been shown. The results of this study have been rationalized by the “ligand exclusion theory.” which has been proposed for the regulation of the glutamic dehydrogenase. It has been shown that the active site and the ADP-binding effector site are oriented close to each other on the enzyme. Furthermore, the data suggest that the adenylic site is not identical to the nonactive coenzyme binding site. A mechanism based on electrostatic interactions is suggested for the cooperative binding of oxidized coenzyme and substrate. Dissociation constants for complexes between the enzyme and two coenzyme fragments (P-ADPR and 2′,5′-ADP) have been estimated.

  • 32.
    Brodelius, Peter
    et al.
    UNIV LUND, CTR CHEM, DEPT PURE & APPL BIOCHEM, S-22007 LUND 7, SWEDEN .
    Lannom, R A
    Kaplan, N O
    The Synthesis of 8-(6-Aminohexyl)-Amino-GMP and Its Applications as a General Ligand in Affinity Chromatography1978In: Archives of Biochemistry and Biophysics, Vol. 188, no 1, 228-231 p.Article in journal (Refereed)
    Abstract [en]

    The synthesis of a new general ligand, i.e., 8-(6-aminohexyl)-amino-GMP, has been achieved by bromination of GMP and subsequent substitution of the bromine for hexamethylene diamine. The overall yield of the synthesis has been 60 to 70%. This new general ligand was immobilized on BrN-activated Sepharose and used as an affinity adsorbent for inosinic acid dehydrogenase (E.C. 1.2.1.14) from Aerobacter aerogenes. Various elution methods were investigated in order to increase the specific activity of the purified enzyme. 

  • 33.
    Brodelius, Peter
    et al.
    Avdelningen for Biokemi, Kemicentrum, Lunds Universitet.
    Larsson, P-O
    Avdelningen for Biokemi, Kemicentrum, Lunds Universitet.
    Mosbach, Klaus
    Avdelningen for Biokemi, Kemicentrum, Lunds Universitet.
    The Synthesis of Three AMP-Analogues: N6-(6-Amino-hexyl)-Adenosine 5'-Monophosphate, N6-(6-Aminohexyl)-Adenosine 2',5'-Bisphosphate, and N6-(6-Aminohexyl)-Adenosine 3',5'-Bisphosphate and Their Application as General Ligands in Biospecific Affinity Chromatography1974In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 47, 81-89 p.Article in journal (Refereed)
    Abstract [en]

    Phosphorylation of 6-chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6-chloropurine-riboside 2’,5‘-bisphosphate and 6-chloropurine-riboside 3‘,5‘-bisphosphate. Reaction with Iy6-diaminohexane followed by resolution of the isomeric mixture on a Dowex 1-X2 column yielded N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 3’,5‘-bisphosphate.The inhibition of several NADP+-dependent and NAD+-dependent dehydrogenases by N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, N6-(6-aminohexyl)-adenosine 3’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 5’-monophosphate was examined.These three AMP-analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P)+-dependent enzymes were investigated. NADP+-dependent enzymes were bound to Sepharose substituted with N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, whereas NAD+-dependent enzymes were not bound under the same conditions. Conversely, when N6-(6-aminohexy1)-adenosine 5‘-monophosphate was used as the immobilised ligand only the NAD+- dependent enzymes were bound, as well as glucose-6-phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group-specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADPf-dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.

  • 34.
    Brodelius, Peter
    et al.
    UNIV LUND, CTR CHEM, DEPT PURE & APPL BIOCHEM, S-22007 LUND 7, SWEDEN .
    Mosbach, K
    Determination of Dissociation Constants for Binary Dehydrogenase-Coenzyme Complexes by (Bio)Affinity Chromatography on an Immobilized AMP-Analogue1976In: Analytical biochemistry, Vol. 72, no 1-2, 629-636 p.Article in journal (Refereed)
    Abstract [en]

    Various alcohol and lactate dehydrogenases were adsorbed to an affinity column of immobilized N6-(6-aminohexyl)-AMP and subsequently eluted with gradients of coenzymes or coenzyme fragments. A linear relation was observed between the eluting concentration of nucleotide and the reported dissociation constants for the corresponding binary enzyme-nucleotide complexes. This relation has been utilized to determine unknown dissociation constants by affinity chromatography. The method presented can also be utilized for the estimation of dissociation constants betweendehydrogenases and coenzyme analogues. 

  • 35.
    Brodelius, Peter
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Mosbach, K
    The Utilization of Immobilized Substrate/Product in Affinity Chromatography. A Model Study using alfa-Chymotrypsin1973In: Acta chemica Scandinavica, Vol. 27, 2634-2638 p.Article in journal (Refereed)
  • 36.
    Brodelius, Peter
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Mosbach, Klaus
    Separation of the Isoenzymes of Lactate Dehydrogenase by Affinity Chromatography Using an Immobilized AMP-Analogue1973In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 35, no 2, 223-226 p.Article in journal (Refereed)
  • 37.
    Brodelius, Peter
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Nilsson, K
    Entrapment of Plant Cells in Different Matrices. A Comparative Study1980In: FEBS letters, Vol. 122, no 2, 312-316 p.Article in journal (Refereed)
  • 38.
    Brodelius, Peter
    et al.
    Lunds Universitet.
    Nilsson, K
    Permeabilization of Immobilized Plant Cells, Resulting in Release of Intracellularly Stored Products with Preserved Cell Viability1983In: European Journal of Applied Microbiology and Biotechnology, ISSN 0340-2118, Vol. 17, no 5, 275-280 p.Article in journal (Refereed)
  • 39.
    Brodelius, Peter
    et al.
    Lunds Universitet.
    Nilsson, K
    Mosbach, K
    Production of alfa-Keto Acids. Part I - Immobilized Cells of Trigonopsis variabilis containing D-Amino Acid Oxidase1981In: Applied biochemistry and biotechnology, Vol. 6, 293-308 p.Article in journal (Refereed)
  • 40.
    Brodelius, Peter
    et al.
    Institute of Biotechnology, Swiss Federal Institute of Technology, Honggerberg, CH-8093 Zurich, Switzerland.
    Vogel, H J
    A Phosphorus-31 Nuclear Magnetic Resonance Study of Phosphate Uptake and Storage in Cultured Catharanthus roseus and Daucus carota Plant Cells1985In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 260, 3556-3560 p.Article in journal (Refereed)
    Abstract [en]

    High resolution "P NMR spectra (103.2 MHz) ofoxygenated Catharanthus roseu8 and Daucus carotacells grown in suspension cultures were obtained usinga solenoidal perfusion probe. The spectra showed resonancesfor various phosphorylated metabolites suchas ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose,and sugar phosphates. The relative levels ofthe phosphorylated metabolites remained constantthroughout the growth curve. No resonances for storagecompounds such as polyphosphates, pyrophosphate,or phytates were observed. Two resolved resonancesfor Pi indicated an intracellular pH of 7.3 and5.7 (or below) for the cytoplasm and vacuoles, respectively.The time course of Pi uptake and storage duringgrowth in fresh culture medium was followed by studyingthe level of vacuolar Pi with 31PN MR (145.7 MHz).Simultaneously, the level of Pi in the culture mediumwas followed with radioactive s2P. C. roseus quicklytakes up all the Pi from the culture medium (maximumrate 1.7 pmol min" g" (dry weight of cells)). The Pi isfirst stored in the vacuoles; subsequently, one part ofthis pool is used to keep a constant cytoplasmic Pi levelwhile another part is apparently accumulated as anNMR invisible Pi store, probably in another cell organelle.In contrast, D. carota does not accumulate Pi inthe vacuoles and consequently it takes up Pi from themedium at a much slower rate (0.05 pmol min" g"(dry weight of cells)). 

  • 41.
    Bustin, Stephen A.
    et al.
    Anglia Ruskin University, UK.
    Nicholls, Ian A.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Iba, Michael
    Rutgers University, USA.
    International Journal of Molecular Science Best Paper Award 20142014In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 15, no 1, 1683-1685 p.Article in journal (Other academic)
  • 42. Carlenor, E
    et al.
    Persson, Bengt L.
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm.
    Glaser, E
    Andersson, B
    Rydström, J
    On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber1988In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 88, 303-308 p.Article in journal (Refereed)
    Abstract [en]

    Mitochondria isolated from potato (Solanum tuberosum L.) tuber wereinvestigated for the presence of a nicotinamide nucleotide transhydrogenaseactivity. Submitochondrial particles derived from these mitochondriaby sonication catalyzed a reduction of NAD' or 3-acetylpyridine-NAD'by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute. milligram protein at pH 5 to 6. The Km values for 3-acetylpyridine-NAD' and NADPH were about 24 and 55 micromolar, respectively.Intact mitochondria showed a negligible activity in the absence of detergents.However, in the presence of detergents the specific activity approachedabout 30% of that seen with submitochondrial particles. Thepotato mitochondria transhydrogenase activity was sensitive to trypsinand phenylarsine oxide, both agents that are known to inhibit the mammaliantranshydrogenase. Antibodies raised against rat liver transhydrogenasecrossreacted with two peptides in potato tuber mitochondrialmembranes with a molecular mass of 100 to 115 kilodaltons. Theobserved transhydrogenase activities may be due to an unspecific activityof dehydrogenases and/or to a genuine transhydrogenase. The activitycontributions by NADH dehydrogenases and transhydrogenase to thetotal transhydrogenase activity were investigated by determining theirrelative sensitivities to trypsin. It is concluded that, at high or neutralpH, the observed transhydrogenase activity in potato tuber submitochondrialparticles is due to the presence of a genuine and specific highmolecular weight transhydrogenase. At low pH an unspecific reaction ofan NADH dehydrogenase with NADPH contributes to the total transhydrogenaseactivity. 

  • 43.
    Carlsson, Stina K.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Effects of adenosine and acetylcholine on the lacrimal gland2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A balanced tear film is essential for a healthy ocular surface. Insufficient tear production may result in dry eye, a common disorder in the elderly population. Dry eye causes significant discomfort in the patients and may lead to visual impairment and ocular infections. The lacrimal gland secretes water, proteins and electrolytes to the aqueous layer of the tear film. Lacrimal gland secretion is tightly regulated by e.g. neuronally released acetylcholine. The effect of acetylcholine on lacrimal gland secretion was recently found to be potentiated by adenosine. Adenosine is an important signaling molecule acting upon the adenosine receptors: A1, A2A, A2B and A3.

    The aim of this thesis was to study effects of adenosine and acetylcholine on intracellular signaling pathways and lacrimal gland secretion. Cholinergic stimulation of secretion was shown to be regulated by the mitogen activated protein kinase p38, a protein previously not known to be involved in exocrine secretion. p38 was activated in response to cholinergic stimulation and inhibition of p38 significantly diminished cholinergic secretion.

    When investigating adenosine effects, potentiation of cholinergic secretion was observed by activation of the A2B receptor in addition to the previously studied A1 receptor. An A2 receptor agonist increased cholinergic rabbit lacrimal gland protein secretion at several concentrations. The increase was inhibited by antagonism of the A2B receptor, but not the A2A receptor. When investigating the intracellular signaling pathways following adenosine and acetylcholine receptor activation, adenosine was shown to increase of cAMP levels. An additional increase in cAMP levels was observed after parallel adenosine and cholinergic receptor activation. Inhibition of Ca2+ release from the endoplasmic reticulum had inhibitory effects of cholinergic stimulation of secretion. In addition, the expression of adenosine receptors in a mouse model of autoimmune dry eye was investigated. The results showed a lymphocyte dependent upregulation of A2A receptors in diseased mice compared to controls.

    In conclusion, the results in this thesis provide significant contributions in the search of dry eye therapeutics through studies of adenosine and acetylcholine receptor activation.

  • 44.
    Chapman, Joanne R.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Waldenström, Jonas
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, e0141853Article, review/survey (Refereed)
    Abstract [en]

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with beta-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  • 45.
    Chavan, Swapnil
    et al.
    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. Uppsala University.
    Karlsson, Björn C. G.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rosengren, Annika M.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ballabio, Davide
    University of Milano-Bicocca, Italy.
    Consonni, Viviana
    University of Milano-Bicocca, Italy.
    Todeschini, Roberto
    University of Milano-Bicocca, Italy.
    Towards Global QSAR Model Building for Acute Toxicity: Munro Database Case Study2014In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 15, no 10, 18162-18174 p.Article in journal (Refereed)
    Abstract [en]

    A series of 436 Munro database chemicals were studied with respect to their corresponding experimental LD50 values to investigate the possibility of establishing a global QSAR model for acute toxicity. Dragon molecular descriptors were used for the QSAR model development and genetic algorithms were used to select descriptors better correlated with toxicity data. Toxic values were discretized in a qualitative class on the basis of the Globally Harmonized Scheme: the 436 chemicals were divided into 3 classes based on their experimental LD50 values: highly toxic, intermediate toxic and low to non-toxic. The k-nearest neighbor (k-NN) classification method was calibrated on 25 molecular descriptors and gave a non-error rate (NER) equal to 0.66 and 0.57 for internal and external prediction sets, respectively. Even if the classification performances are not optimal, the subsequent analysis of the selected descriptors and their relationship with toxicity levels constitute a step towards the development of a global QSAR model for acute toxicity.

  • 46.
    Clima, Rosanna
    et al.
    Univ Bari,Italy ; Univ Bologna, Italy.
    Preste, Roberto
    Univ Bari, Italy.
    Calabrese, Claudia
    European Bioinformat Inst EMBL Outstn Hinxton, UK.
    Diroma, Maria Angela
    Univ Bari, Italy.
    Santorsola, Mariangela
    Univ Bari, Italy.
    Scioscia, Gaetano
    IBM Italia SpA, Italy ; MBLab Bari, Italy.
    Simone, Domenico
    Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
    Shen, Lishuang
    Childrens Hosp Los Angeles, USA.
    Gasparre, Giuseppe
    Univ Bologna, Italy.
    Attimonelli, Marcella
    Univ Bari, Italy.
    HmtDB 2016: data update, a better performing query system and human mitochondrial DNA haplogroup predictor2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no D1, D698-D706 p.Article in journal (Refereed)
    Abstract [en]

    The HmtDB resource hosts a database of human mitochondrial genome sequences from individuals with healthy and disease phenotypes. The database is intended to support both population geneticists as well as clinicians undertaking the task to assess the pathogenicity of specific mtDNA mutations. The wide application of next-generation sequencing (NGS) has provided an enormous volume of high-resolution data at a low price, increasing the availability of human mitochondrial sequencing data, which called for a cogent and significant expansion of HmtDB data content that has more than tripled in the current release. We here describe additional novel features, including: (i) a complete, user-friendly restyling of the web interface, (ii) links to the command-line stand-alone and web versions of the MToolBox package, an up-to-date tool to reconstruct and analyze human mitochondrial DNA from NGS data and (iii) the implementation of the Reconstructed Sapiens Reference Sequence (RSRS) as mitochondrial reference sequence. The overall update renders HmtDB an even more handy and useful resource as it enables a more rapid data access, processing and analysis. HmtDB is accessible at http://www.hmtdb.uniba.it/.

  • 47. Clyne, N
    et al.
    Persson, Bengt L.
    Department of Biochemistry, University of Stockholm.
    Havu, N
    Hultman, E
    Lins, L-E
    Pehrsson, S K
    Rydström, J
    Wibom, R
    The intracellular distribution of cobalt in exposed and unexposed rat myocardium1990In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 50, no 6, 605-609 p.Article in journal (Refereed)
    Abstract [en]

    The intracellular distribution of cobalt was analysed in the myocardium of exposed and unexposed rats. The exposed rats were given a dietary cobalt supplementation of 40 mg CoS04-7 H20/kg body weight for 8 weeks. The mitochondrial fraction showed the greatest relative increase in cobalt: 0.09 ng/mg protein in the unexposed rats to 8.43 ng/mg protein in the exposed rats. In the exposed rats the submitochondrial particles had the highest levels of cobalt: 19.43 ng/mg protein, followed by the sarcoplasmatic reticulum: 12.3 ng/mg protein. The microsomal 44 000g supernatant also showed an increase, although the levels remained low (0.51 ng/mg protein in the exposed animals). Apparently the calcium-storing organelles had the highest levels of cobalt. This could affect calcium flux in myocardial cells and, secondarily, tension development in cardiac muscle.

  • 48. Collinge, M A
    et al.
    Brodelius, Peter
    Institute of Biotechnology ETH-Hönggerberg CH-8093 Zürich, Switzerland.
    Dynamics of Benzophenanthridine Alkaloid Production in Suspension Cultures of Eschscholtzia californica after Treatment with a Yeast Elicitor1989In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 28, no 4, 1101-1104 p.Article in journal (Refereed)
    Abstract [en]

    A number of benzophenanthridine alkaloids are induced in suspension cultures of Eschscholtzia californica after treatment with an elicitor prepared from yeast extract. The formation of the alkaloids sanguinarine, chelerythrine and macarpine has been studied in relation to; elicitor concentration, incubation time after elicitation, and culture age. A significant portion of these alkaloids is released into the medium. Sanguinarine and chelerythrine reach maximum levels a few hours after the time of elicitation. Thereafter, their levels decline and the amount of macarpine increases. Viability of elicited cells, as determined by their subsequent growth, is not significantly reduced. There is a good correlation between induced tyrosine decarboxylase activity and alkaloid formation. 

  • 49.
    Crona, Mikael
    et al.
    Arrhenius Laboratories for Natural Sciences.
    Avesson, Lotta
    Swedish University of Agricultural Sciences.
    Sahlin, Margareta
    Arrhenius Laboratories for Natural Sciences ; Stockholm University.
    Lundin, Daniel
    Stockholm University.
    Hinas, Andrea
    Swedish University of Agricultural Sciences.
    Klose, Ralph
    Arrhenius Laboratories for Natural Sciences.
    Söderbom, Fredrik
    Swedish University of Agricultural Sciences.
    Sjöberg, Britt-Marie
    Arrhenius Laboratories for Natural Sciences ; Swedish University of Agricultural Sciences.
    A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 12, 8198-8208 p.Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.

  • 50.
    Daly, Norelle
    et al.
    University of Queensland, Australia.
    Chen, Yi
    University of Queensland, Australia.
    Rosengren, K. Johan
    University of Queensland, Australia.
    Marx, Ute
    University of Queensland, Australia.
    Phillips, Martin
    UCLA, USA.
    Waring, Alan
    UCLA, USA.
    Wang, Wei
    UCLA, USA.
    Lehrer, Robert
    UCLA, USA.
    Craik, David
    University of Queensland, Australia.
    Retrocyclin-2: a potent anti-HIV theta-defensin that forms a cyclic cysteine ladder structural motif2009In: Peptides for Youth: The Proceedings of the 20th American Peptide Symposium / [ed] Valle S.D., Escher E., Lubell W.D., Springer, 2009, Vol. 611, no 11, 577-578 p.Conference paper (Refereed)
12345 1 - 50 of 206
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