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  • 1.
    Abellan-Flos, Marta
    et al.
    Univ Namur, Belgium;PSL Univ, France.
    Timmer, Brian J. J.
    KTH Royal Institute of Technology, Sweden.
    Altun, Samuel
    Attana AB, Sweden.
    Aastrup, Teodor
    Attana AB, Sweden.
    Vincent, Stephane P.
    Univ Namur, Belgium.
    Ramström, Olof
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. KTH Royal Institute of Technology, Sweden;Univ Massachusetts, USA.
    QCM sensing of multivalent interactions between lectins and well-defined glycosylated nanoplatforms2019In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 139, article id 111328Article in journal (Refereed)
    Abstract [en]

    Quartz crystal microbalance (QCM) methodology has been adopted to unravel important factors contributing to the "cluster glycoside effect" observed in carbohydrate-lectin interactions. Well-defined, glycosylated nanostructures of precise sizes, geometries and functionalization patterns were designed and synthesized, and applied to analysis of the interaction kinetics and thermodynamics with immobilized lectins. The nanostructures were based on Borromean rings, dodecaamine cages, and fullerenes, each of which carrying a defined number of carbohydrate ligands at precise locations. The synthesis of the Borromeates and dodecaamine cages was easily adjustable due to the modular assembly of the structures, resulting in variations in presentation mode. The binding properties of the glycosylated nanoplatforms were evaluated using flow-through QCM technology, as well as hemagglutination inhibition assays, and compared with dodecaglycosylated fullerenes and a monovalent reference. With the QCM setup, the association and dissociation rate constants and the associated equilibrium constants of the interactions could be estimated, and the results used to delineate the multivalency effects of the lectin-nanostructure interactions.

  • 2.
    Acuña, Ulyana Muñoz
    et al.
    Ohio State University, USA.
    Curley, Robert W
    Ohio State University, USA.
    Fatima, Nighat
    Quaid-i-Azam University, Pakistan;University of Hawaii at Hilo, USA.
    Ahmed, Safia
    Quaid-i-Azam University, Pakistan.
    Chang, Leng Chee
    University of Hawaii at Hilo, USA.
    De Blanco, Esperanza J Carcache
    Ohio State University, USA.
    Differential Effect of Wortmannolone Derivatives on MDA-MB-231 Breast Cancer Cells.2017In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 37, no 4, p. 1617-1623Article in journal (Refereed)
    Abstract [en]

    BACKGROUND/AIM: The survival rate of women diagnosed with triple-negative breast-cancer (TNBC) remains low. Hence, this study aimed at the chemical and biological optimization of furanosteroid derivatives for the treatment of this type of malignancy using TNBC cells.

    MATERIALS AND METHODS: Semi-synthetic analogs of wortmannolone (1-6) that negatively affected the aberrant pathways in tumor cells were evaluated in hormone-independent breast cancer cells using western blot and cell-cycle analysis.

    RESULTS: Wortmannolone derivatization generated NF-ĸB inhibitors as new lead structures for further development. Compound (3) was found to be the most significantly active lead.

    CONCLUSION: Structure-activity analysis in the present study showed that acetylation of the hydroxyl groups and substitution on C3 and C17 of wortmannolone enhanced biological activity. Alpha-substitution of the acetyl group in C3 on ring A (compound 3) resulted in ROS inducing effect; however, presence of an acetyl group in β-position of C3 displayed the highest NF-ĸB p65 inhibitory activity (0.60 μM).

  • 3.
    Adler, Anna
    et al.
    Uppsala University, Sweden.
    Fritsch, Marlene
    Uppsala University, Sweden.
    Fromell, Karin
    Uppsala University, Sweden.
    Leneweit, Gero
    ABNOBA GmbH, Germany;Assoc Promot Canc Therapy, Germany.
    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
    Uppsala University, Sweden;Cellular & Mol Biotechnol Res Inst CMB, Japan;Univ Tsukuba, Japan.
    Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro2023In: Journal of materials chemistry. B, ISSN 2050-750X, E-ISSN 2050-7518, Vol. 11, no 46, p. 11121-11134Article in journal (Refereed)
    Abstract [en]

    Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 degree celsius. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

  • 4.
    Adler, Anna
    et al.
    Uppsala University, Sweden.
    Inoue, Yuuki
    The University of Tokyo, Japan.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Baba, Teruhiko
    National Institute of Advanced Industrial Science and Technology, Japan.
    Ishihara, Kazuhiko
    The University of Tokyo, Japan.
    Nilsson, Bo
    Uppsala University, Sweden.
    Teramura, Yuji
    Uppsala University, Sweden;National Institute of Advanced Industrial Science and Technology, Japan.
    Effect of liposome surface modification with water-soluble phospholipid polymer chain-conjugated lipids on interaction with human plasma proteins2022In: Journal of materials chemistry. B, ISSN 2050-750X, E-ISSN 2050-7518, Vol. 10, no 14, p. 2512-2522Article in journal (Refereed)
    Abstract [en]

    Alternative liposome surface coatings for PEGylation to evade the immune system, particularly the complement system, have garnered significant interest. We previously reported poly(2-methacryloyloxyethyl phosphorylcholine) (MPC)-based lipids (PMPC-lipids) and investigated the surface modification of liposomes. In this study, we synthesize PMPC-lipids with polymerization degrees of 10 (MPC10-lipid), 20 (MPC20-lipid), 50 (MPC50-lipid), and 100 (MPC100-lipid), and coated liposomes with 1, 5, or 10 mol% PMPC-lipids (PMPC-liposomes). Non-modified and PEGylated liposomes are used as controls. We investigate the liposome size, surface charge, polydispersity index, and adsorption of plasma proteins to the liposomes post incubation in human plasma containing N,N,N′,N′-ethylenediamine tetraacetic acid (EDTA) or lepirudin by some methods such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and automated capillary western blot, with emphasis on the binding of complement protein C3. It is shown that the coating of liposome PMPC-lipids can suppress protein adsorption more effectively with an increase in the molecular weight and molar ratio (1-10 mol%). Apolipoprotein A-I is detected on PMPC-liposomes with a higher molecular weight and higher molar ratio of PMPC-lipids, whereas α2-macroglobulin is detected on non-modified, PEGylated, and PMPC-liposomes with a shorter polymer chain. In addition, a correlation is shown among the PMPC molecular weight, molar ratio, and C3 binding. The MPC10-lipid cannot inhibit C3 binding efficiently, whereas surface modifications with 10 mol% MPC20-lipid and 5 mol% and 10 mol% MPC50-lipid suppress both total protein and C3 binding. Hence, liposome modification with PMPC-lipids can be a possible strategy for avoiding complement activation.

  • 5.
    Adler, Anna
    et al.
    Uppsala University, Sweden.
    Inoue, Yuuki
    Univ Tokyo, Japan.
    Sato, Yuya
    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. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials. Uppsala University, Sweden.
    Nilsson, Bo
    Uppsala University, Sweden.
    Teramura, Yuji
    Uppsala University, Sweden;Univ Tokyo, Japan;Natl Inst Adv Ind Sci & Technol, Japan.
    Synthesis of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated lipids and their characterization and surface properties of modified liposomes for protein interactions2021In: Biomaterials Science, ISSN 2047-4830, E-ISSN 2047-4849, Vol. 9, no 17, p. 5854-5867Article in journal (Refereed)
    Abstract [en]

    Poly(ethylene glycol) (PEG) is frequently used for liposomal surface modification. However, as PEGylated liposomes are cleared rapidly from circulation upon repeated injections, substitutes of PEG are being sought. We focused on a water-soluble polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units, and synthesized poly(MPC) (PMPC)-conjugated lipid (PMPC-lipid) with degrees of MPC polymerization ranging from 10 to 100 (calculated molecular weight: 3 to 30 kDa). In addition, lipids with three different alkyl chains, myristoyl, palmitoyl, and stearoyl, were applied for liposomal surface coating. We studied the interactions of PMPC-lipids with plasma albumin, human complement protein C3 and fibrinogen using a quartz crystal microbalance with energy dissipation, and found that adsorption of albumin, C3 and fibrinogen could be suppressed by coating with PMPC-lipids. In particular, the effect was more pronounced for PMPC chains with higher molecular weight. We evaluated the size, polydispersity index, surface charge, and membrane fluidity of the PMPC-lipid-modified liposomes. We found that the effect of the coating on the dispersion stability was maintained over a long period (98 days). Furthermore, we also demonstrated that the anti-PEG antibody did not interact with PMPC-lipids. Thus, our findings suggest that PMPC-lipids can be used for liposomal coating.

  • 6.
    Ahlstrand, Emma
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Metal ions in life: towards accurate computer-aided studies ofprotein-ion interactions2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The importance of ions in life sciences can not be overstated. The interaction betweenmetal ions and proteins is vital because it is involved in a variety of biological processes.The ions contribute to stability and function of proteins. Moreover, they are relevant indisease progression.Realistic computer simulations pave the way for drug development, through providingdetailed insights into the dynamics of proteins and various biological processes thatoccur in the body. Such information can be impossible to achieve through experimentsof living subjects in vivo or from test tube experiments in vitro alone. However,theoretical methods have to result in accurate predictions. In my thesis, I studieddifferent ways to handle the ions in simulations. Since the systems contain thousands ofatoms the calculations are demanding. Despite the availability of computer clusters, thecom putational capacity is not sufficient. I have examined the simplified models used insimulations of larger systems (e.g., whole proteins) to pave the way for improvements ofthe simulation models.Different ions have different effects on biochemical systems and it is important to beable to distinguish between them. Thus, from a biochemical point of view, it is centralto be able to describe their unique characteristics. Their difference can be from vital totoxic to the body. Zinc is essential and present in more than 3000 proteins in our bodyand has a very flexible interaction with proteins. This property has proved to be hard toreproduce in computer simulations. Cadmium can replace zinc, but is toxic because itdoes not have the same catalytic ability. From a modelling perspective do these ions havesimilar characteristics as they have the same ionic charge. Inclusion of more realisticelectron effects may be necessary to be able to simulate the difference.With my studies, I have contributed towards a better understanding of the interactionsbetween metal ions and proteins. I have pointed out a direction for further improvementof methods for simulations of large systems.For the same purpose, I have also studied the frequently occurring ions sodium andpotassium found as salts in all body fluids, but also lithium belonging to the same groupin the periodic table and used in therapeutic purposes. The results show that potassiumand sodium can be simulated by a commonly used computational approach, whereasmore advanced methods are required to study lithium ions accurately.Overall, the work within this thesis has explored ion-protein interactions and providedinformation about methods for energy calculations and models for molecular dynamicssimulations for some of the most important ions within biochemistry.

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  • 7.
    Alstermark, Mirjam
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Fluorescens in situ hybridisering: Optimering och vidareutveckling av en kurslaboration på Biomedicinska analytikerprogrammet2019Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Fluorescens in situ hybridization (FISH) is used to detect cytogenetic aberrations and abnormalities of Deoxyribonucleic acid (DNA) and Ribonucleic Acid (RNA). The FISH begins with chromosome extraction of the desired cell preparation then a direct or indirect fluorescently labeled probe (15-30 base pair long) is hybridized to its genetic target sequence. The preparation can thereafter be analyzed in fluorescence microscope to see bound probe at chromosome level. In the course “Advanced laboratory methodology” for the Biomedical Scientist program, Linnaeus University, a FISH laboratory experiment is conducted where results have not been clear nor reproducible.

    The aim of this study was to improve the laboratory experiment FISH.

    Human Cardiac Microvascular Endothelial Cells (HCMEC) was grown to 60 % and 80 % confluence, to an estimated number of ≥ 3 x106, and analyzed by G-band staining and DNA-FISH. G-band staining showed many cells in interphase and few free chromosomes of cells with 60 % confluence. G-band staining and DNA-FISH showed that cells grown to 80 % confluence showed more free chromosomes from metaphase. The cancer cell lines VMM1 and H1915 were therefore grown to 80 % confluence and ≥ 3 x106. Multicolor-FISH on VMM1 and H1915 showed results from all painting probes blue/aqua, red and green. The conclusion is that in chromosomal extraction from cultured adherent cells should be 80 % confluent to give clear and reproducible probe staining of chromosomes in metaphase when assayed with Multicolor FISH. Analysis of 80 % confluent cells and the use of Multicolor FISH technology is a clear improvement to the “Advanced laboratory methodology” course.

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  • 8.
    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)
  • 9.
    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)
  • 10.
    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, K. 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, article id 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.

  • 11.
    Andersson, Håkan S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Uppsala university, Sweden.
    Jacobsson, Erik
    Uppsala University, Sweden.
    Laborde, Quentin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Rosengren, K. Johan
    University of Queensland, Australia.
    Strand, Malin
    Swedish University of Agricultural Sciences, Sweden.
    Göransson, Ulf
    Uppsala University, Sweden.
    Alpha-nemertides - a novel family of nemertean peptide neurotoxins2018Conference paper (Other academic)
    Abstract [en]

    We recently discovered a novel family of neuroactive peptides in nemerteans, which we have named alpha-nemertides (1). One of these peptides, nemertide alpha-1, has been the subject of detailed studies with regard to structure and effects. The peptide exhibits exceptional potency against a number of arthropod species. Moreover, in vitro experiments suggest that alpha-1 acts primarily on voltage-gated sodium channels, and that this action is selective for arthropods by two orders of magnitude over vertebrate species. Using transcriptomic and proteomic approaches, we have identified 10 alpha-nemertides, but this number is likely to increase. These peptides alongside with a series of mutants are currently under evaluation by our group, with the goal to improve our understanding of structure-function relationships. In addition, we are considering potential practical uses of alpha-nemertides. In this talk, I will describe the current status of this research project.

    1. E. Jacobsson et al., Peptide ion channel toxins from the bootlace worm, the longest animal on Earth. Scientific reports 8, 4596 (2018).

  • 12.
    Andersson, Håkan S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Jacobsson, Erik
    Uppsala University.
    Rosengren, K. 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.

  • 13.
    Andersson, Håkan S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Jacobsson, Erik
    Uppsala University, Sweden.
    Rosengren, K. Johan
    University of Queensland, Australia.
    Strand, Malin
    Swedish University of Agricultural Sciences, Sweden.
    Göransson, Ulf
    Uppsala University, Sweden.
    Mapping the diversity of nemertean peptide toxins2018Conference paper (Other academic)
  • 14.
    Andersson, Håkan S.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Jacobsson, Erik
    Uppsala University, Sweden.
    Strand, Malin
    Swedish University of Agricultural Sciences, Sweden.
    Peigneur, Steve
    University of Leuven (KU Leuven), Belgium.
    Lebbe, Eline
    University of Leuven (KU Leuven), Belgium.
    Rosengren, K. Johan
    University of Queensland.
    Tytgat, Jan
    University of Leuven (KU Leuven), Belgium.
    Göransson, Ulf
    Uppsala University, Sweden.
    Alpha-nemertides, a novel family of marine peptide neurotoxins from ribbon worms2017Conference paper (Other academic)
  • 15.
    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.

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  • 16.
    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.

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  • 17.
    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, ISSN 0006-3088, E-ISSN 1336-9563, Vol. 67, no 6, p. 1056-1061Article 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.

  • 18.
    Andre, Ingemar
    et al.
    Lund University, Sweden.
    Bjelic, Sinisa
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Computational assessment of folding energy landscapes in heterodimeric coiled coils2018In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 86, no 7, p. 790-801Article in journal (Refereed)
    Abstract [en]

    The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs-into-holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.

  • 19.
    Ardeshirilajimi, Sheida
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Non-enzymatic activation ofC5 in the tumor microenvironment2021Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    The complement system is an elegant biochemical cascade consisting of around 50serum soluble and membrane-bound proteins that defend the body from pathogens. It is a central part of the innate immune system, and it is regarded as a first-line defence mechanism of the human body against invading pathogens. The complement system can be activated via three pathways, alternative pathway, classical pathway, and lectin pathway. In all three pathways, C5 convertases are produced to cleave C5 into C5band C5a for host defence. In some environments, such as low pH, pro-oxidative and pro-inflammatory environment; instead, C5 can be activated without any cleavage of the C5 but confronted with a conformational change and expresses a C5b-like structure, still containing the C5a-domain bound to the main molecule. In contrast to normal cells, cancer cells prefer to aspirate in the anaerobic pathway even though there is enough oxygen, so they produce lactic acid, which creates an acidic environment in the tumor microenvironment (TME). The TME is further pro-oxidative and pro-inflammatory, which promotes tumor progression. This thesis aim was to simulate the TME ex vivo to understand the in vivo physiological role of non-enzymatic activation of C5. We hoped to answer whether C5b-like, which existence in vivo has never been proved, was generated in the TME or not.C5 faced a conformational change when serum was acidified with HCl or lactic acid. In a pro-oxidative environment with serum-supplemented with H2O2, a low amount of C5b-like was detected, but by adding iron catalyzer FeCl2, this amount was increased. With adding gallic acid as an antioxidant, the C5 conformational change was increased even further. Hence, C5 non-enzymatic activation in the presence ofH2O2 was iron-dependent, and by adding antioxidants, the turnover of C5b-like increased. In addition, C5b-like was also detected when activating granulocytes in serum. The C5 conformational change was also investigated in conditioned medium from the tumor cell line BXPC-3, an approach to mimic the TME with low pH and pro-oxidative status. BXPC-3 cells produced lactate in cell culture media and C5blikewas produced in the media by adding the serum as a C5 source. In conclusion, we show that a C5 conformational change occurred in low pH, in a prooxidative, and in a pro-inflammatory environment, which are the three characteristics of the TME. Complement activation in the TME may therefore progress without the controlled formation of C5-convertases.

  • 20.
    Asawa, Kenta
    et al.
    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. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials. Uppsala University, Sweden.
    Nilsson, Bo
    Uppsala University, Sweden.
    Teramura, Yuji
    Univ Tokyo, Japan;Uppsala University, Sweden.
    Cell Surface Functionalization with Heparin-Conjugated Lipid to Suppress Blood Activation2021In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 31, no 11, article id 2008167Article in journal (Refereed)
    Abstract [en]

    Organ transplantation leads to damage of the endothelial glycocalyx of the transplanted organ, and the activated endothelial surface induces thromboinflammation. The result is dysfunction of the transplanted organ, known as ischemia reperfusion injury (IRI). Long-term graft survival strongly depends on the regulation of IRI. Here the aim is to reconstruct the glycocalyx to regulate blood activation during IRI. Heparin-conjugated lipid (fHep-lipid) is synthesized with 0.6, 1.8, 2.7, 4.5, or 8.0 fragmented heparins per lipid to compare their anticoagulation activity. First, liposome and cells are modified with each fHep-lipid and the surface properties are evaluated. Then the hemocompatibility of the modified human mesenchymal stem cells (hMSCs) is examined in a loop model using human blood. The antithrombin-binding capacity and anti-factor Xa activity of the fHep-lipids depend on the number of conjugated heparins, with efficacy increasing with increasing number of heparins. The modified liposomes are highly negatively charged and show strong anti-factor Xa activity. In addition, the cell surfaces of human erythrocytes and hMSCs can be uniformly modified with fHep-lipid. The whole blood studies reveal that fHep-lipid on hMSCs can prevent generation of thrombin-antithrombin complexes, coagulation markers, and platelet aggregation, whereas unmodified hMSCs trigger activation of the platelet and coagulation systems.

  • 21.
    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, p. S87-S87Article in journal (Other academic)
  • 22.
    Ataya, Mohammad Kher
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Varför binder dasatinib mycket bra till vissa proteiner men mindre bra till andra?2020Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Introduction: Cancer is a group of about 200 different types of diseases. Cancer can appear in different cells and tissues in the body when abnormal cells grow in an uncontrolled manner and invade other tissues. A multistep process that occurs over a long period eventually leads to the onset of cancer. Kinases are enzymes that catalyze phosphorylation, that is, the reaction in which a phosphate group from ATP is transferred to another molecule. Kinase phosphorylation is an important mechanism for modifying proteins and thus regulating various cellular functions. Overactivity of kinases can lead to loss of control of signal transducer cascades controlled by kinases and it produces harmful effects such as cardiovascular disease, diabetes and cancer. Chronic myeloid leukemia (CML) is a rare blood cancer in which an abnormal BCR-ABL fusion protein occurs in neoplastic cells. This causes leukocytes to be produced abnormally and damages the bone marrow and blood. Dasatinib is a tyrosine kinase inhibitor indicated for the treatment of CML. Dasatinib inhibits the activity of the BCR-ABL kinase, but since many kinases are similar, it also binds to other kinases. Some of the kinases that dasatinib inhibits and that we analyzed in this study are ABL1, PTK6, MYT1 and STK10.

    Purpose: The purpose of this study was to explain with the help of the structures of the proteins ABL1, PTK6, MYT1 and STK10 why dasatinib binds well to some proteins but less well to others.

    Methods: Using the structures of the proteins, an analysis of interactions between dasatinib and the selected proteins has been performed. We then built a model for each protein with only the amino acids that binds to the drug and finally used these structures to estimate the binding energy ΔGb with the PM7 theoretical method. The PM7 method was used to calculate the energy of molecules. The results correspond to the enthalpy of formation ΔfH. The Gibbs energy is estimated by including the solvent, which also accounts for entropy changes, most of which are mediated by the solvent. The equation used to calculate Gibbs energy for protein-drug binding was: ΔGb = ΔfG(complex) - [ΔfG(drug) + ΔfG(protein)].

    Results: The results show that there is a link between the number of dasatinib-protein interactions and Kd. Some proteins had several different structures in the same crystal. In such cases there was an extensive variation in the number of interactions between the different structures and dasatinib. The connection between the number of interactions and Kd was not perfect. Using the number of interactions we were able to distinguish between the proteins that bind best to dasatinib, which were ABL1 and PTK6, and the proteins that bind worst which were MYT1 and STK10. We could however not explain which protein binds best based on the number of interactions. The theoretical calculation of ΔGb follows the same trend (the more interactions there are in the structures, the lower is ΔGb).

    Discussion and conclusion: A relatively quick and easy calculation using a built model (dasatinib with protein binding interaction) could be used to distinguish between the best and worst binders. When it comes to the difference between ABL1 and PTK6, our analysis failed. A large difference between the calculated ΔGb values ​​for different structures of the same protein makes it difficult to choose the right structure for an analysis. A factor that could explain the discrepancy in the analysis is that the structure of the protein changes when it binds to the drug and such a change requires an investment of Gibbs energy. In general, experimental binding energies were higher than the calculated ones. For example, experimental ΔGb for MYT1 was -9 kcal/mol but the calculated ΔGb was -33.2 kcal/mol. This may be because the calculation with PM7 is too simplified.

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  • 23.
    Azuma, Tomoyuki
    et al.
    Univ Tokyo, Japan.
    Matsushita, Taishi
    Univ Tokyo, Japan.
    Manivel, Vivek Anand
    Uppsala University, Sweden.
    Nilsson Ekdahl, Kristina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Linnaeus University, Linnaeus Knowledge Environments, Advanced Materials. Uppsala University, Sweden.
    Nilsson, Bo
    Uppsala University, Sweden.
    Teramura, Yuji
    Univ Tokyo, Japan;Uppsala University, Sweden.
    Takai, Madoka
    Univ Tokyo, Japan.
    Poly(2-aminoethyl methacrylate)-based polyampholyte brush surface with carboxylic groups to improve blood compatibility2020In: Journal of Biomaterials Science. Polymer Edition, ISSN 0920-5063, E-ISSN 1568-5624, Vol. 31, no 5, p. 679-693Article in journal (Refereed)
    Abstract [en]

    Zwitterionic material-based polymer brush significantly prevents protein adsorption and cell adhesion, which leads to the blood compatibility. However, zwitterionic polymer itself is difficult to be modified further, for the blood compatibility since the charged balance is impaired after the modification. In this research, chemically modifiable mixed charge polymer brush is designed, without impairing its characteristics. Condensed mixed charge polymer brush will work like zwitterionic material because neighbouring opposite charge is reported to be important in the zwitterionic material. Cationic polymer brush with primary amine group, which is based on 2-aminoethyl methacrylate (AEMA), was prepared and modified by succinic anhydride to obtain carboxylic group induced poly(AEMA). The ratio of primary amine group and carboxylic group was optimized to obtain the polyampholyte brush. The blood compatibility was evaluated by measuring coagulation/complement activation, protein adsorption and cell adhesion induced by the polymer. Our designed cationic-based polyampholyte brush prevented coagulation/complement activation comparable to poly(2-methacryloyloxyethyl phosphorylcholine) brush, based on intra-monomer interaction, because condensed mix charge works like zwitterion.

  • 24.
    Bacovic, Betim
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Validering och tillämpning av en bioassay baserad på Artemia salina för undersökning av giftverkan: Experimentell studie2019Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Background: Alpha-nemertides are about 30 aminoacids long so-called ICK peptides, which have been shown to be very toxic to shore crabs and a number of other invertebrates. A survey project where the presence of alpha-nematides in marine so-called ribbon worms has been investigated for four years as a collaboration between researchers at the Linnaeus University, Uppsala University, the Swedish Agricultural University and the University of Queensland, Brisbane. The nature and toxicity of these peptides have been investigated by several methods, including a bioassay based on Artemia salina, for which a method previously has been developed. The task of this work was to test the method in order to be able to use it in a quality-assured way in the laboratory at Linnaeus University.

    Aim/Method: The aim of this study was to develop and validate a reliable protocol for estimating acute toxicity in shore crabs using juveniles from Artemia Salina (brine shrimp) in microtiter plates.

    Results:  Results showed that the artemia assay was reliable. Results between the series A1, B1 and C1 showed the same IC50. The emetin studies showed, compared to previous studies, a devation of about a ten-potency, however, EC50 for alpha-1 and alpha-2 matched previous studies from Uppsala.

    Discussion/Conclusion: Assay can be used for comparison with corresponding trials in Uppsala. Further attempts are necessary to clarify within which timeframes to check the microwell plates. 

  • 25. 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, p. 261-266Article in journal (Refereed)
  • 26.
    Bengtsson, Elina
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Cooperativity in muscle proteins: a study of actin filaments and myosin II2014Doctoral thesis, comprehensive summary (Other academic)
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  • 27.
    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, p. 299A-300A, article id 1499-PosArticle in journal (Other academic)
  • 28.
    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, p. 718-728Article 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.

  • 29.
    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, p. 1465-1477Article 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.

  • 30.
    Berg, Albin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Identifiering av housekeeping-gener för att studera genuttryck hos Synechococcus odlade med eller utan kisel2021Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    To study gene expressions in an organism, it is necessary to use housekeeping genes. Approved housekeeping genes can be defined as genes with a constant gene expression independent of silica concentration. A good housekeeping gene makes it possible to reliable study changes in gene expression in other functional genes that may have a role in silica uptake in Synechococcus. Housekeeping genes therefore act as an internal standard. Synechococcus can take up silica but the physiological function and how silica get transported into Synechococcus is still unknown. The aim of this thesis is to see if the potential housekeeping genes in Synechococcus remain constant in present and absence of silica. The goal is that those housekeeping genes later can be used for quantitative reverse transcription polymerase chain reaction (RT-qPCR) to study other functional genes in Synechococcus. In this study, 11 different primer pairs were designed to bind to four different genes (ppc, rnpA, rnpB and secA) in the Synechococcus genome. Results from PCR and gel electrophoresis shows that several of these primers bind complementarily to the organism's DNA. The isolates that gave amplified product were selected to be further worked with and RNA extraction was performed on one single Synechococcus isolate. The Synechococcus isolate was grown in the presence of silica and without silica. RT-qPCR synthesis were performed on this isolate with the Sec_A_3-primer pair with varying RNA concentrations and a positive DNA control. The result from RT-qPCR shows lack of -amplified product despite the PCR showing that the Sec_A_3 primer gave specific amplified products against some of the isolates. The RT-qPCR method failed to be optimized and the DNA and RNA-samples gave no amplification. Troubles with optimizing the RT-qPCR method made it impossible to study the RNA expression of Synechococcus in the presence and absence of silica. Despite failed RT-qPCR and qPCR, this study contributes with several potential primer sequences that can be used against several different Synechococcus isolates. If the methods get optimized the four housekeeping genes ppc, rnpA, rnpB and secA may in the future be used to study the expression of other functional genes in Synechococcus.

  • 31.
    Berg, Albin
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Towards single molecule actin-activated myosin ATPase assay for rapid screening of myosin mutants2023Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The development of miniaturized assays for the actin based molecular motors of the myosin type, is crucial to reduce the workload and time to extract important information associated with the chemomechanical cycle of the motor. It is also important with such assays to better understand a plethora of associated myopathy-causing mutations. In previous studies, single molecule methods involving TIRF microscopy have been established to study the basal myosin ATPase activity. The aim of this study was to further upgrade the single molecule assay to be able to extract the corresponding actin-activated myosin ATPase activity. Inspired by previous studies, crosslinking of human β-cardiac myosin on top of surface-immobilized F-actin was performed using the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). The results suggest promising crosslinking efficiency of myosin on top of F-actin. Myosin molecules thus persisted on the filaments despite the introduction of ATP that otherwise dissociates the actomyosin complex. Three different methods of immobilizing F-actin to a nitrocellulose surface were investigated, biotin-streptavidin-, NEM-HMM-based and direct crosslinking of F-actin to nitrocellulose. Overall crosslinking of F-actin to nitrocellulose shows reliable immobilization of filaments while the other methods examined have their advantages and disadvantages. The results suggest that further optimization is required for the biotin-streptavidin and NEM-HMM based attachment. Using the developed assay, we were able to detect binding of fluorescently labelled ATP to myosin, cross-linked on top of F-actin, at single molecule resolution.

  • 32.
    Berggren, Gustav
    et al.
    Uppsala University.
    Lundin, Daniel
    Stockholm University.
    Sjöberg, Britt‐Marie
    Stockholm University.
    Assembly of Dimanganese and Heterometallic Manganese Proteins2017In: Encyclopedia of Inorganic and Bioinorganic Chemistry / [ed] Robert A. Scott, John Wiley & Sons, 2017Chapter in book (Refereed)
  • 33.
    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, p. 171-184Article 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.

  • 34.
    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, p. 132-141Article 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.

  • 35.
    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, p. 177-177Article in journal (Other academic)
  • 36.
    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, p. 14521-14528Article 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.

  • 37.
    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, p. 10049-10057Article 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.

  • 38.
    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, p. 256-271Article 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.

  • 39.
    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, p. 2285-2305Article 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.

  • 40.
    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, p. 749-757Article 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.

  • 41.
    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, p. 7709-7723Article 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.

  • 42. 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, p. 859-868Article 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. 

  • 43. 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, p. 452-457Article 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. 

  • 44.
    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, p. 23-29Article 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. 

  • 45.
    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, p. 219-223Article in journal (Refereed)
  • 46.
    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, p. 561-566Article 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. 

  • 47.
    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, p. 280-285Article 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. 

  • 48.
    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, p. 93-97Article in journal (Refereed)
  • 49.
    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, p. 2651-2654Article 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. 

  • 50.
    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, p. 186-188Article 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. 

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