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  • 251.
    Tobieson, Lovisa
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Neurokirurgiska kliniken US.
    Ghafouri, Bijar
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för samhällsmedicin. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Smärt och rehabiliteringscentrum.
    Zsigmond, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Neurokirurgiska kliniken US.
    Rossitti, Sandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Neurokirurgiska kliniken US.
    Hillman, Jan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Sinnescentrum, Neurokirurgiska kliniken US.
    Marklund, Niklas
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Lund Univ, Sweden.
    Dynamic protein changes in the perihaemorrhagic zone of Surgically Treated Intracerebral Haemorrhage Patients2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 3181Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The secondary injury cascades exacerbating the initial brain injury following intracerebral haemorrhage (ICH) are incompletely understood. We used dual microdialysis (MD) catheters placed in the perihaemorrhagic zone (PHZ) and in seemingly normal cortex (SNX) at time of surgical ICH evacuation in ten patients (range 26-70 years). Routine interstitial MD markers (including glucose and the lactate/pyruvate ratio) were analysed and remaining microdialysate was analysed by two-dimensional gel electrophoresis (2-DE) and nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS). Two time intervals were analysed; median 2-10 hours post-surgery (time A) and median 68-76 hours post-ICH onset (time B). Using 2-DE, we quantified 232 +/- 31 different protein spots. Two proteins differed between the MD catheters at time A, and 12 proteins at time B (p amp;lt; 0.05). Thirteen proteins were significantly altered between time A and time B in the SNX and seven proteins in the PHZ, respectively. Using nLC-MS/MS ca 800 proteins were identified out of which 76 were present in all samples. At time A one protein was upregulated and two downregulated, and at time B, seven proteins were upregulated, and four downregulated in the PHZ compared to the SNX. Microdialysis-based proteomics is feasible for study of secondary injury mechanisms and discovery of biomarkers after ICH.

  • 252.
    Tress, Wolfgang
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska fakulteten.
    Organic Solar Cells: Theory, Experiment, and Device Simulation2014Bok (Refereegranskat)
    Abstract [en]

    This book covers in a textbook-like fashion the basics or organic solar cells, addressing the limits of photovoltaic energy conversion and giving a well-illustrated introduction to molecular electronics with focus on the working principle and characterization of organic solar cells. Further chapters based on the author’s dissertation focus on the electrical processes in organic solar cells by presenting a detailed drift-diffusion approach to describe exciton separation and charge-carrier transport and extraction. The results, although elaborated on small-molecule solar cells and with focus on the zinc phthalocyanine: C60 material system, are of general nature. They propose and demonstrate experimental approaches for getting a deeper understanding of the dominating processes in amorphous thin-film based solar cells in general.

     The main focus is on the interpretation of the current-voltage characteristics (J-V curve). This very standard measurement technique for a solar cell reflects the electrical processes in the device. Comparing experimental to simulation data, the author discusses the reasons for S-Shaped J-V curves, the role of charge carrier mobilities and energy barriers at interfaces, the dominating recombination mechanisms, the charge carrier generation profile, and other efficiency-limiting processes in organic solar cells. The book concludes with an illustrative guideline on how to identify reasons for changes in the J-V curve.

     This book is a suitable introduction for students in engineering, physics, material science, and chemistry starting in the field of organic or hybrid thin-film photovoltaics. It is just as valuable for professionals and experimentalists who analyze solar cell devices.

  • 253.
    Tu, William B.
    et al.
    University of Toronto, Canada; Princess Margaret Cancer Centre, Canada.
    Helander, Sara
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Filosofiska fakulteten. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Pilstål, Robert
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska fakulteten.
    Ashley Hickman, K.
    University of Toronto, Canada; Princess Margaret Cancer Centre, Canada.
    Lourenco, Corey
    University of Toronto, Canada; Princess Margaret Cancer Centre, Canada.
    Jurisica, Igor
    University of Toronto, Canada; Princess Margaret Cancer Centre, Canada.
    Raught, Brian
    University of Toronto, Canada; Princess Margaret Cancer Centre, Canada.
    Wallner, Björn
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska fakulteten.
    Sunnerhagen, Maria
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Penn, Linda Z.
    University of Toronto, Canada; Princess Margaret Cancer Centre, Canada.
    Myc and its interactors take shape2015Ingår i: Biochimica et Biophysica Acta. Gene Regulatory Mechanisms, ISSN 1874-9399, E-ISSN 1876-4320, Vol. 1849, nr 5, s. 469-483Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    The Myc oncoprotein is a key contributor to the development of many human cancers. As such, understanding its molecular activities and biological functions has been a field of active research since its discovery more than three decades ago. Genome-wide studies have revealed Myc to be a global regulator of gene expression. The identification of its DNA-binding partner protein, Max, launched an area of extensive research into both the protein-protein interactions and protein structure of Myc. In this review, we highlight key insights with respect to Myc interactors and protein structure that contribute to the understanding of Mycs roles in transcriptional regulation and cancer. Structural analyses of Myc show many critical regions with transient structures that mediate protein interactions and biological functions. Interactors, such as Max, TRRAP, and PTEF-b, provide mechanistic insight into Mycs transcriptional activities, while others, such as ubiquitin ligases, regulate the Myc protein itself. It is appreciated that Myc possesses a large interactome, yet the functional relevance of many interactors remains unknown. Here, we discuss future research trends that embrace advances in genome-wide and proteome-wide approaches to systematically elucidate mechanisms of Myc action. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. (C) 2014 Elsevier B.V. All rights reserved.

  • 254.
    Turkina, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Functional proteomics of protein phosphorylation in algal photosynthetic membranes2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in the permanently changing natural environment. For successful survival and growth photosynthetic organisms have developed complex sensing and signaling acclimation mechanisms. The environmentally dependent protein phosphorylation in photosynthetic membranes is implied in the adaptive responses; however, the molecular mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected to distinct environmental conditions known to affect the photosynthetic machinery.

    The state transitions process regulating the energy distribution between two photosystems, involves the temporal functional coupling of phosphorylated light-harvesting complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work provided evidences suggesting that redox-dependent phosphorylation-induced structural changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may uncouple this protein together with LHCII from both photosystems to minimize the damaging effects of excess light.

    Reversible phosphorylation of the PSII reaction center proteins was shown to be essential for the maintenance of active PSII under high light stress. Particularly dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII reaction center, is required for its degradation and replacement. We found in the alga the reversible D1 protein phosphorylation, which until our work, has been considered as plant-specific.

    We also discovered specific induction of thylakoid protein phosphorylation during adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2- and redox-dependent protein phosphorylation may be an early adaptive and signalling response of the green alga to limitation in inorganic carbon.

    This work provides the first comprehensive insight into the network of environmentally regulated protein phosphorylation in algal photosynthetic membranes.

    Delarbeten
    1. CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii
    Öppna denna publikation i ny flik eller fönster >>CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii
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    2006 (Engelska)Ingår i: Proteomics, ISSN 1615-9853, Vol. 6, nr 9, s. 2693-2704Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.

    Nyckelord
    Amino acid sequencing, Chlamydomonas reinhardtii, CO2 limitation, Mass spectrometry, Protein phosphorylation
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-12927 (URN)10.1002/pmic.200500461 (DOI)
    Tillgänglig från: 2008-02-06 Skapad: 2008-02-06 Senast uppdaterad: 2009-06-05
    2. The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation
    Öppna denna publikation i ny flik eller fönster >>The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation
    2004 (Engelska)Ingår i: FEBS letters, ISSN 0014-5793, Vol. 564, nr 1-2, s. 104-108Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The surface-exposed peptides were cleaved by trypsin from the photosynthetic thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. Two phosphorylated peptides, enriched from the peptide mixture and sequenced by nanospray quadrupole time-of-flight mass spectrometry, revealed overlapping sequences corresponding to the N-terminus of a nuclear-encoded chlorophyll a/b-binding protein CP29. In contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in the mature algal CP29 was not removed but processed by methionine excision, N-terminal acetylation and phosphorylation on threonine 6. The importance of this phosphorylation site is proposed as the reason of the unique transit peptide retention.

    Nyckelord
    Transit peptide, Thylakoid membrane, CP29, Protein phosphorylation, Mass spectrometry, Chlamydomonas reinhardtii
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-12928 (URN)10.1016/S0014-5793(04)00323-0 (DOI)
    Tillgänglig från: 2008-02-06 Skapad: 2008-02-06 Senast uppdaterad: 2009-06-05
    3. Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions
    Öppna denna publikation i ny flik eller fönster >>Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions
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    2005 (Engelska)Ingår i: The FEBS journal, ISSN 1742-464X, Vol. 272, nr 18, s. 4797-4806Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI–PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.

    Nyckelord
    Chlamydomonas, CP29, photosynthesis, protein phosphorylation, State transitions
    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-12929 (URN)10.1111/j.1742-4658.2005.04894.x (DOI)
    Tillgänglig från: 2008-02-06 Skapad: 2008-02-06
    4. Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii
    Öppna denna publikation i ny flik eller fönster >>Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii
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    2006 (Engelska)Ingår i: Molecular & cellular proteomics, ISSN 1535-9476, Vol. 5, nr 8, s. 1412-1425Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-12930 (URN)10.1074/mcp.M600066-MCP200 (DOI)
    Tillgänglig från: 2008-02-06 Skapad: 2008-02-06 Senast uppdaterad: 2009-06-05
  • 255.
    Turner, Anthony P.F.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biosensorer och bioelektronik. Linköpings universitet, Tekniska fakulteten.
    Paper potentiostats for bioelectrochemistry2015Ingår i: Program of the XXIII International Symposium on Bioelectrochemistry and Bioenergetics of the Bioelectrochemical Society. 14-18 June, 2015. Malmö, Sweden, Lausenne: Bioelectrochemical Society , 2015, s. 166-166Konferensbidrag (Övrigt vetenskapligt)
    Abstract [en]

    This presentation will focus on meeting current challenges in decentralised diagnostics by using amperometric and votammetric systems printed on paper or plastic substrates to deliver inexpensive instruments for a wide range of electroanalytical applications. This approach combines the sophistication of advanced electrochemical biosensors with a simple manufacturing technique to create a use-and-throw instrument. A key driver for next-generation electroanalytical devices is rapid, convenient and easy ways to measure our body chemistries at the genomic, proteomic and metabolomic levels. We are targeting fully-integrated platforms such as all-printed biosensing systems, integrated sampling and wearable devices. Further development will result in cost reduction and a diversity of formats such as point-of-care tests, smart packaging, telemetric paper strips and print-on-demand analytical devices. These platforms form workhorses in our hands for a variety of diagnostic systems including enzyme electrodes for multi-parametric diabetes monitoring and for the management of chronic kidney disease, electrochemical sensors for enzymes such as G6-P or amylase (a marker for stress), label-free affinity sensors for cancer markers and heart disease, aptasensors for cancer cells, DNA Sensors and robust devices based on imprinted and smart polymers. Using these technologies, we envision over-the-counter paper instruments for self-diagnosis of common diseases such as diabetes, kidney disease and urinary tract infection; inexpensive devices for use by caregivers or paramedics such as the ”Stressometer” or heart attack indicators; home kits to support people after transplant surgery or cancer treatment, smart cartons for pharmaceuticals; pocket tests for allergens, food toxicity, drinking water etc. and strips or patches that communicate with mobile telecommunications. Realisation of these paradigm-changing new products requires the effective harnessing of emerging technology, inspired vision from clinical partners or others “users” and leading-edge engineering to design and produce functional systems in appropriate volumes at the right cost.

  • 256.
    Ungerback, Jonas
    et al.
    CALTECH, CA 91125 USA; Lund Univ, Sweden.
    Hosokawa, Hiroyuki
    CALTECH, CA 91125 USA.
    Wang, Xun
    CALTECH, CA 91125 USA.
    Strid, Tobias
    Lund Univ, Sweden.
    Williams, Brian A.
    CALTECH, CA 91125 USA.
    Sigvardsson, Mikael
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för mikrobiologi och molekylär medicin. Linköpings universitet, Medicinska fakulteten. Lund Univ, Sweden.
    Rothenberg, Ellen V
    CALTECH, CA 91125 USA.
    Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPIT (PU.1)2018Ingår i: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 28, nr 10, s. 1508-1519Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    SPI1 (also known as PU.1) is a dominant but transient regulator in early T-cell precursors and a potent transcriptional controller of developmentally important pro-T-cell genes. Before T-lineage commitment, open chromatin is frequently occupied by PU.1, and many PU.1 sites lose accessibility when PU.1 is later down-regulated. Pioneering activity of PU.1 was tested in this developmentally dynamic context by quantitating the relationships between PU.1 occupancy and site quality and accessibility as PU.1 levels naturally declined in pro-T-cell development and by using stage-specific gain- and loss-offunction perturbations to relate binding to effects on target genes. PU.1 could bind closed genomic sites, but rapidly opened many of them, despite the absence of its frequent collaborator, CEBPA. RUNX motifs and RUNX1 binding were often linked to PU.1 at open sites, but highly expressed PU.1 could bind its sites without RUNX1 The dynamic properties of PU.1 engagements implied that PU.1 binding affinity and concentration determine its occupancy choices, but with quantitative trade-offs for occupancy between site sequence quality and stage-dependent site accessibility in chromatin. At nonpromoter sites, PU.1 binding criteria were more stringent than at promoters, and PU.1 was also much more effective as a transcriptional regulator at non promoter sites where local chromatin accessibility depended on the presence of PU.1. Notably, closed chromatin presented a qualitative barrier to occupancy by the PU.1 DNA-binding domain alone. Thus, effective pioneering at closed chromatin sites also depends on requirements beyond site recognition, served by non-DNA-binding domains of PU.1.

  • 257.
    Urbancic, Dunja
    et al.
    Univ Ljubljana, Slovenia.
    Kotar, Anita
    Slovenian NMR Ctr, Slovenia.
    Smid, Alenka
    Univ Ljubljana, Slovenia.
    Jukic, Marko
    Univ Ljubljana, Slovenia.
    Gobec, Stanislav
    Univ Ljubljana, Slovenia.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Plavec, Janez
    Slovenian NMR Ctr, Slovenia.
    Mlinaric-Rascan, Irena
    Univ Ljubljana, Slovenia.
    Methylation of selenocysteine catalysed by thiopurine S-methyltransferase2019Ingår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1863, nr 1, s. 182-190Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown. Methods: To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and Se-77 NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations. Results: Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by Se-77 NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy. Conclusions: The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner. General significance: Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid.

  • 258.
    Utterström, Johanna
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik.
    One Bead One Compound Screening for Cyclic Peptide Binding Partners2018Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    In recent years a significant research focus has been on the development of biomimicking three-dimensional substrates for cell culturing. Hydrogels mimicking the extracellular matrix is a well-suited scaffold for this purpose and there are many different ways these can be cross-linked to retain their shape. The group of Molecular Materials at IFM, Linköping University, is focusing on the development of physical hydrogels hybridized through peptide-peptide interactions but all peptides used for this today are created using rational design and on top of this very large, making them time-consuming and expensive to fabricate. The aim of this project was to evaluate if One Bead One Compound (OBOC) libraries could be used as an alternative to rational design in the finding of cyclic peptide binding partners used in the hybridization of hydrogels. The results were not very promising though since only seven peptides passed all screening steps and of these only two could be sequenced. Of these two, only one was water soluble enough to enable binding interactions analysis but was then found to be a false hit. Nevertheless, it should be noticed that only a fraction of all possible combinations was screened and the results cannot exclude OBOC libraries as an approach in the quest of finding new cyclic peptide binding partners.

  • 259.
    Vainonen, Julia P
    et al.
    University of Turku, Turku, Finland.
    Vener, Alexander
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Aro, Eva-Mari
    University of Turku, Turku, Finland.
    Determination of in vivo Protein Phosphorylation in Photosynthetic Membranes2009Ingår i: Plant Signal Transduction: Methods and Protocols / [ed] Thomas Pfannschmidt, New York, NY, United States: Humana Press, 2009, Vol. 479, s. 133-146Kapitel i bok, del av antologi (Övrigt vetenskapligt)
    Abstract [en]

    Light- and redox-controlled reversible phosphorylation of thylakoid proteins regulates short- and long-term acclimation of plants to environmental cues. The major phosphoproteins in thylakoids belong to photosystem II and its light-harvesting antenna but phosphorylation of subunits of other thylakoid protein complexes has been detected as well. The detection methods include electrophoretic separation of proteins and detection of phosphoproteins with a phosphoaminoacid-specific antibody or phosphoprotein-specific dye. The use of mass spectrometry allows the identification of exact phosphorylation site(s) in the proteins. Various methods for detection of phosphoproteins in thylakoids are outlined including phosphopeptide preparation for mass spectrometric analyses and quantitative analysis of protein phosphorylation.

  • 260.
    Veenstra, Cynthia
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    The receptor tyrosine kinase Met and the protein tyrosine phosphatase PTPN2 in breast cancer2017Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Breast cancer is the most common form of cancer in women worldwide and the second leading cause of cancer death. It is a heterogeneous disease and is subdivided into different subtypes, all with different treatment responses and survival outcomes. Luminal breast cancers are characterised by the expression of oestrogen receptor and generally have a good prognosis. More aggressive tumours are marked by the presence of growth stimulating receptor tyrosine kinase HER2 (HER2-like breast cancer) or the absence of oestrogen receptor, progesterone receptor, and HER2 (triple-negative breast cancer,TNBC). The latter is the most aggressive form and is difficult to treat due to lack of treatment targets.

    This thesis aimed to explore possible prognostic and predictive biomarkers in different subtypes and study their role in breast cancer. To this aid, breast cancer tumours of pre- and post-menopausal patients enrolled in two cohorts were analysed for gene copy numbers and expression of proteins involved in cell proliferation. Gene copy numbers of receptor tyrosine kinases MET and EGFR, Met’s ligand HGF, and protein tyrosine phosphatase PTPN2 were determined by droplet digital PCR or quantitative PCR in both cohorts. Met, phosphorylated Met (pMet), HGF, and PTPN2 protein expression levels were analysed with immunohistochemical staining in the pre-menopausal cohort. Moreover,the role of the aforementioned proteins was investigated in breast cancer cell lines. Amplification of MET, HGF, and EGFR in breast tissues was found to be low (5-8%). These three genes, all located on chromosome 7, were found to be strongly correlated with eachother and to be associated with shortened distant recurrence-free survival. High protein expression of Met, pMet, and HGF was found in 33%, 53%, and 49% of the breast tumours. MET and EGFR were found to be more often amplified in TNBC disease, correlating with worse survival. Moreover, stromal expression of HGF was associated with shorter survival in TNBC. EGF stimulation in TNBC cell line MDA-MB-468 led to inhibited cell proliferation and migration. Partial knockdown of EGFR caused TNBC cells to proliferate and migrate more upon EGF treatment, mirroring EGFR inhibitor resistance. Knockdown of Met had in part the opposite effects, indicating that Met inhibitors might be useful in the treatment of TNBC. The increase in proliferation and migration upon EGFR depletion could be counteracted with simultaneous knockdown of EGFR and Met, indicating that dual inhibition of these proteins might be a future treatment option in TNBC.

    Copy loss of PTPN2 was reported in 15% of the cases in both pre- and post-menopausal cohorts. Low cytoplasmic PTPN2 protein expression was found in half of the cases. Loss of PTPN2 gene or protein was associated with a shorter distant recurrence-free survival in Luminal A and HER2-positive tumours, not in TNBC, suggesting a subtype-related prognostic value of PTPN2. Subtype relevance of PTPN2 was further implied by in vitro analyses. Whereas PTPN2 knockdown had no observed effect on TNBC cell lines, knockdown in the Luminal A cell line MCF7 inhibited Met phosphorylation and promoted phosphorylation of Akt, a key regulator of cellular proliferation and survival. The cell growth and survival regulating RAS/MAPK pathway remained unaffected. Knockdown in the HER2-positive cell line SKBR3 led to increased Met phosphorylation and decreased RAS/MAPK-related Erk phosphorylation as well as EGF-mediated transcription factor STAT3 phosphorylation. These results indicate that the role of PTPN2 in breast cancer is subtype-related and needs to be further investigated for future treatment options.

    Delarbeten
    1. Met and its ligand HGF are associated with clinical outcome in breast cancer
    Öppna denna publikation i ny flik eller fönster >>Met and its ligand HGF are associated with clinical outcome in breast cancer
    Visa övriga...
    2016 (Engelska)Ingår i: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, nr 24, s. 37145-37159Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Few biomarkers exist to predict radiotherapy response in breast cancer. In vitro studies suggest a role for Met and its ligand HGF. To study this suggested role, MET and HGF gene copy numbers were determined by droplet digital PCR in tumours from 205 pre-menopausal and 184 post-menopausal patients, both cohorts randomised to receive either chemo-or radiotherapy. MET amplification was found in 8% of the patients in both cohorts and HGF amplification in 7% and 6% of the patients in the pre-and post-menopausal cohort, respectively. Met, phosphorylated Met (pMet), and HGF protein expression was determined by immunohistochemistry in the premenopausal cohort. Met, pMet, and HGF was expressed in 33%, 53%, and 49% of the tumours, respectively. MET amplification was associated with increased risk of distant recurrence for patients receiving chemotherapy. For the pre-menopausal patients, expression of cytoplasmic pMet and HGF significantly predicted benefit from radiotherapy in terms of loco-regional recurrence. Similar trends were seen for MET and HGF copy gain. In the post-menopausal cohort, no significant association of benefit from radiotherapy with neither genes nor proteins was found. The present results do not support that inhibition of Met prior to radiotherapy would be favourable for pre-menopausal breast cancer, as previously suggested.

    Ort, förlag, år, upplaga, sidor
    IMPACT JOURNALS LLC, 2016
    Nyckelord
    radiation; copy number variation; droplet digital PCR; triple-negative breast cancer; radiotherapy
    Nationell ämneskategori
    Cancer och onkologi
    Identifikatorer
    urn:nbn:se:liu:diva-130130 (URN)10.18632/oncotarget.9268 (DOI)000377756800127 ()27175600 (PubMedID)
    Anmärkning

    Funding Agencies|Swedish Cancer Society; Swedish Research Council; LiU Cancer Foundation

    Tillgänglig från: 2016-07-12 Skapad: 2016-07-11 Senast uppdaterad: 2019-06-28
  • 261.
    Veibäck, Axel
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Development of an expression system for a dehydrogenase2010Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [sv]

    Biokatalytiska processteg har de senaste åren blivit ett allt viktigare inslag i kemisk syntes för att åstadkomma ekonomisk och miljövänlig produktion. För att utvärdera användandet av enzymer i en process hos Cambrex Karlskoga AB utvecklades ett expressionssystem för ett dehydrogenas. En syntetisk gen klonades in i Escherichia coli DH5a och uttrycktes med hjälp av expressionsvektorn pTZ19R, som tidigare finns beskrivet i litteraturen. Proteinuttrycket utfördes vid 25°C, 30°C och 37°C och resultatet mättes med hjälp av SDS-PAGE och aktivitetsmätningar. Genen för dehydrogenaset modifierades på tre sätt, vilket gav upphov till åtta varianter. Genen fördes över till expressionsvektorn pSE420, kortades för att undvika bildning av inklusionskroppar och en nukleotid som fattades från gensekvensen återinfördes. Ett protokoll utarbetades även för undersökning av inklusionskroppar. Till sist sammanställdes en metod för att undersöka de kinetiska konstanterna hos dehydrogenaset. Slutsatsen av arbetet är att fortsatta studier måste utföras för att erhålla uttryck av dehydrogenaset och rekommendationer ges för framtida undersökningar.

     

  • 262.
    Vicente Carrillo, Alejandro
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för kliniska vetenskaper. Linköpings universitet, Medicinska fakulteten.
    Sperm Membrane Channels, Receptors and Kinematics: Using boar spermatozoa for drug toxicity screening2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.

    Delarbeten
    1. Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa
    Öppna denna publikation i ny flik eller fönster >>Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But Not of Aquaporin 9 in Boar Spermatozoa
    2016 (Engelska)Ingår i: Reproduction in domestic animals, ISSN 0936-6768, E-ISSN 1439-0531, Vol. 51, nr 5, s. 665-679Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen-thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP-7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid-piece and principal piece domains in ejaculated spermatozoa. AQP-9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP-7, but not that of AQP-9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP-9 but relocated AQP-7 towards the acrosome. AQP-7, but not AQP-9, appears as a relevant marker for non-empirical studies of sperm handling.

    Ort, förlag, år, upplaga, sidor
    Blackwell Verlag, 2016
    Nationell ämneskategori
    Utvecklingsbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-130228 (URN)10.1111/rda.12728 (DOI)000388334100005 ()27405395 (PubMedID)
    Anmärkning

    Funding agencies: Swedish Research Council VR, Stockholm [521-2011-6553]; Research Council FORMAS, Stockholm [221-2011-512]; FORSS (Forskningsradet i Sydostra Sverige), Sweden [473121]

    Tillgänglig från: 2016-07-19 Skapad: 2016-07-19 Senast uppdaterad: 2018-03-23Bibliografiskt granskad
    2. The CatSper channel modulates boar sperm motility during capacitation.
    Öppna denna publikation i ny flik eller fönster >>The CatSper channel modulates boar sperm motility during capacitation.
    2017 (Engelska)Ingår i: Reproductive biology, ISSN 2300-732X, Vol. 17, nr 1, s. 69-78Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The cation channel of sperm (CatSper) comprises four transmembrane subunits specifically expressed in human, equine, murine and ovine spermatozoa, apparently implicated in capacitation, hyperactivation and acrosome exocytosis. Western blotting and immunocytochemistry showed hereby that CatSper subunits are also present in boar spermatozoa, primarily over the sperm neck, tail and cytoplasmic droplets; albeit CatSper -1 presented in addition some distribution over the membrane of the acrosome and CatSper -2 and -4 over the membrane of the post-acrosome. The role of the Catsper channel in boar spermatozoa was investigated by extending the spermatozoa in media containing different calcium (Ca(2+)) availability and exposure to the capacitation-trigger bicarbonate, to progesterone or CatSper inhibitors (Mibefradil and NNC 55-0396), separately or sequentially, at physiological and toxicological doses. Extracellular Ca(2+) availability, combined with bicarbonate exposure (capacitation-inducing conditions) decreased sperm motility, similarly to when spermatozoa incubated in capacitation-inducing conditions was exposed to Mibefradil and NNC 55-0396. Exposure of these spermatozoa to progesterone did not cause significant changes in sperm motility and nor did it revert its decrease induced by CatSper antagonists. In conclusion, the CatSper channel regulates sperm motility during porcine capacitation-related events in vitro.

    Ort, förlag, år, upplaga, sidor
    Elsevier, 2017
    Nyckelord
    CatSper, Spermatozoa, Capacitation, Sperm motility, Boar
    Nationell ämneskategori
    Biologiska vetenskaper
    Identifikatorer
    urn:nbn:se:liu:diva-134398 (URN)10.1016/j.repbio.2017.01.001 (DOI)000397370900009 ()28077244 (PubMedID)2-s2.0-85009212156 (Scopus ID)
    Anmärkning

    Funding agencies: The Swedish Research council VR, Stockholm [521-2011-6553]; Research Council FORMAS, Stockholm [221-2011-512]; FORSS (Forskningsradet i Sydostra Sverige, Sweden [473121]

    Tillgänglig från: 2017-02-23 Skapad: 2017-02-23 Senast uppdaterad: 2017-05-29Bibliografiskt granskad
    3. The mu (µ) and delta (δ) opioid receptors modulate boar sperm motility
    Öppna denna publikation i ny flik eller fönster >>The mu (µ) and delta (δ) opioid receptors modulate boar sperm motility
    2016 (Engelska)Ingår i: Molecular Reproduction and Development, ISSN 1040-452X, E-ISSN 1098-2795, Vol. 83, nr 8, s. 724-734Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Endogenous and exogenous opioids modulate reproductive functions in target cells via opioid receptors (µ, δ, and κ). Sperm motility is a metric of gamete functionality, and serves as a suitable parameter for in vitro drug-induced toxicity assays. This study identifies the presence and location of opioid receptors in pig spermatozoa as well as their functional response after in vitro challenge with known agonists (morphine [µ]; [D-Pen 2,5]-enkephanile [δ]; and U 50488 [κ]) and antagonists (naloxone [µ]; naltrindole [δ]; and nor-binaltrorphimine [κ]). Only the µ- and δ-opioid receptors were present in the sperm plasma membrane, overlying the acrosome, neck, and principal piece. Challenge experiments with agonists and antagonists identified both µ- and δ-opioid receptors as regulators of sperm kinematics, wherein µ maintains or increases sperm movement whereas δ decreases sperm motility over time. This article is protected by copyright. All rights reserved.

    Ort, förlag, år, upplaga, sidor
    John Wiley & Sons, 2016
    Nyckelord
    opioids, membrane receptors, kinematics, pig
    Nationell ämneskategori
    Utvecklingsbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-130181 (URN)10.1002/mrd.22675 (DOI)000387014800007 ()27391529 (PubMedID)
    Anmärkning

    Funding agencies: Swedish Research Council VR, Stockholm [521-2011-6553]; Research Council FORMAS, Sweden [221-2011-512]; Research Council in Southeast Sweden (FORSS), Sweden [378091/31297]

    Tillgänglig från: 2016-07-14 Skapad: 2016-07-14 Senast uppdaterad: 2017-11-28Bibliografiskt granskad
    4. Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles
    Öppna denna publikation i ny flik eller fönster >>Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles
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    2015 (Engelska)Ingår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 29, nr 3, s. 582-591Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker and JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P less than 0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r= 0.559) and proportions of motile (r = 0.55) or progressively motile (r = 0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

    Ort, förlag, år, upplaga, sidor
    Elsevier, 2015
    Nyckelord
    Sperm; Motility; Mitochondria; Drug; Toxicity; Boar
    Nationell ämneskategori
    Klinisk medicin
    Identifikatorer
    urn:nbn:se:liu:diva-117655 (URN)10.1016/j.tiv.2015.01.004 (DOI)000352050100019 ()25624015 (PubMedID)
    Anmärkning

    Funding Agencies|Swedish Research Council (VR); Research Council Formas, Stockholm, Sweden

    Tillgänglig från: 2015-05-12 Skapad: 2015-05-06 Senast uppdaterad: 2017-12-04
  • 263.
    Villebeck, Laila
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Structural rearrangements of actins interacting with the Chaperonin systems TRiC/Prefoldin and GroEL/ES2007Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The studies in this thesis are mainly focused on the effects that the chaperonin mechanisms have on a bound target protein. Earlier studies have shown that the bacterial chaperonin GroEL plays an active role in unfolding a target protein during the initial binding. Here, the effects of the eukaryotic chaperonin TRiC’s mechanical action on a bound target protein were studied by fluorescence resonance energy transfer (FRET) measurements by attaching the fluorophore fluorescein to specific positions in the structure of the target protein, β-actin. Actin is an abundant eukaryotic protein and is dependent on TRiC to reach its native state. It was found that at the initial binding to TRiC, the actin structure is stretched, particularly across the nucleotide-binding site. This finding led to the conclusion that the binding-induced unfolding mechanism is conserved through evolution. Further studies indicated that in a subsequent step of the chaperonin cycle, the actin molecule collapses. This collapse leads to rearrangements of the structure at the nucleotide-binding cleft, which is also narrowed as a consequence.

    As a comparison to the productive folding of actin in the TRiC chaperonin system, FRET studies were also performed on actin interacting with GroEL. This is a non-productive interaction in terms of guiding actin to its native state. The study presents data indicating that the nucleotide-binding cleft in actin is not rearranged by GroEL in the same way as it is rearranged during the TRiC interaction. Thus, it could be concluded that although the general unfolding mechanism is conserved through the evolution of the chaperonins, an additional and specific binding to distinct parts of the actin molecule has evolved in TRiC. This specific binding leads to a directed unfolding and rearrangement of the nucleotide-binding cleft, which is vital for actin to reach its native state. The differences in the chemical properties of the actin-GroEL and the actin-TRiC complexes were also determined by measurements of fluorescein anisotropies and AEDANS emission shifts for probes attached to positions spread throughout the actin structure.

    The evolutionary aspects of the chaperonin mechanisms and the target protein binding were further investigated in another study. In this study, the prokaryotic homologue to actin, MreB, was shown to bind to both TRiC and GroEL. MreB was also shown to bind to the co-chaperonin GroES.

    In a separate study, the interaction between actin and the chaperone prefoldin was investigated. In vivo prefoldin interacts with non-native actin and transfers it to TRiC for subsequent and proper folding. In this homo-FRET study, it was shown that actin binds to prefoldin in a stretched conformation, similar to the initial binding of actin to TRiC.

    Delarbeten
    1. Conformational Rearrangements of Tail-less Complex Polypeptide 1 (TCP-1) Ring Complex (TRiC)-Bound Actin
    Öppna denna publikation i ny flik eller fönster >>Conformational Rearrangements of Tail-less Complex Polypeptide 1 (TCP-1) Ring Complex (TRiC)-Bound Actin
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    2007 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, Vol. 46, nr 17, s. 5083-5093Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC−actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-13123 (URN)10.1021/bi062093o (DOI)
    Tillgänglig från: 2008-04-03 Skapad: 2008-04-03 Senast uppdaterad: 2018-04-25
    2. Different Conformational Effects when β-actin Binds to the Bacterial Chaperonin GroEL and the Eukaryotic Chaperonin TRiC
    Öppna denna publikation i ny flik eller fönster >>Different Conformational Effects when β-actin Binds to the Bacterial Chaperonin GroEL and the Eukaryotic Chaperonin TRiC
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    2007 (Engelska)Artikel i tidskrift (Refereegranskat) Submitted
    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-13124 (URN)
    Tillgänglig från: 2008-04-03 Skapad: 2008-04-03 Senast uppdaterad: 2018-04-25
    3. Mapping the Different Interactions between Eukaryotic β-actin and the Group I (GroEL) and Group II (TRiC) Chaperonins
    Öppna denna publikation i ny flik eller fönster >>Mapping the Different Interactions between Eukaryotic β-actin and the Group I (GroEL) and Group II (TRiC) Chaperonins
    Visa övriga...
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Productive folding to the native state of the abundant eukaryotic protein actin is dependent on the chaperonin TRiC. The prokaryotic chaperonin GroEL also recognizes actin, but this interaction does not lead to the correct folding of actin. It is well established that GroEL interacts with non-native proteins through hydrophobic interactions. The characteristics of the interactions between TRiC and its target proteins are however unclear. In this study, we present multiple site-directed cysteine labeling and fluorescence measurements indicating that actin initially binds to TRiC through several interaction sites and that the surfaces of the interaction areas on the walls of the TRiC chamber present both polar and hydrophobic residues. At a later stage in the chaperonin cycle, the binding of ATP causes conformational changes in the chaperonin, which leads to a presentation of a more hydrophobic milieu in TRiC chamber. The conformational changes of the chaperonin causes rearrangements of the actin molecule and new interactions are proposed to be formed. Additionally, we show that the initial binding of actin to TRiC leads to a re-modeling of the nucleotide-binding cleft in actin. We also present data indicating that GroEL presents less specific interaction areas towards the bound actin than TRiC does. The interactions between actin and GroEL are tight and of hydrophobic character. No re-modeling of the nucleotide-binding cleft was obtained in the actin-GroEL complex. We conclude that the interactions between actin and TRiC are of both polar and hydrophobic character, the nature of the interactions are different in the prokaryotic and eukaryotic chaperonins, and the rearrangements of the nucleotide binding cleft of actin seen in the chaperonin cycle of TRiC do not occur in GroEL. We suggest that the rearrangements of the nucleotide-binding site in actin are critical for productive folding of actin.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-52934 (URN)
    Tillgänglig från: 2010-01-13 Skapad: 2010-01-13 Senast uppdaterad: 2018-04-25
    4. Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiC
    Öppna denna publikation i ny flik eller fönster >>Interactions Between the Bacterial β-actin Homologue MreB and the Group I Chaperonin GroEL and Group II Chaperonin TRiC
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    This pilot study on the interaction between MreB andthe chaperonins TRiC and GroEL indicates that thefolding of the actin ancestor was facilitated by thechaperonins. From an evolutionary point of view, it isinteresting to investigate the nature of the bindinginteraction between the prokaryotic system MreB-GroELand compare it to the binding interaction between actinand TRiC and the following questions will be addressed:Does MreB refold in a spontaneous manner or is itsfolding dependent on the action of a chaperonin (GroEL)as in the case of actin folding (TRiC)? Does MreB bind ina similar stretched manner to GroEL as actin binds toTRiC (4, 11), or is the “general” binding inducedunfolding sufficient for guiding MreB to the native state?How does the MreB molecule interact with TRiC, is therea similar stretching as for actin? Are there any analoguessequences between actin and TRiC that are recognized byTRiC and/or GroEL?

    Two single variants where cysteines have beenintroduced at positions 69 and 245 in E. coli MreB(Figure 1 B). These positions are situated at the tips of thecorresponding subdomains 2 and 4 of the actin molecule(4, 12). The double variant N69C/V245C has also beenconstructed. The three variants will be produced andlabeled with fluorescein and subsequent homo-FRETmeasurements will be performed on MreB bound toGroEL, TRiC and GroES. The results will be comparedto the results on actin bound to the chaperonins toinvestigate how the chaperonin-dependent folding ofactin homologues has evolved.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-52935 (URN)
    Tillgänglig från: 2010-01-13 Skapad: 2010-01-13 Senast uppdaterad: 2018-04-25Bibliografiskt granskad
    5. Elongation of Actin Upon Binding to Prefoldin
    Öppna denna publikation i ny flik eller fönster >>Elongation of Actin Upon Binding to Prefoldin
    (Engelska)Manuskript (Övrig (populärvetenskap, debatt))
    Abstract [en]

    This pilot study on the interaction between actin andprefoldin indicates that actin binds to prefoldin in astretched conformation, probably similar to the bindingconformation at the initial interaction with TRiC/ADP as shown in a previous study (8). Further refinement of theprefoldin purification is a future aim, as well as additionalhomo-FRET measurements on several actin variantsspread throughout the structure to investigate the localvolume expansions/compressions of the prefoldin-boundactin molecule and compare the results to actin bound tothe chaperonins TRiC/ADP, TRiC/AMP-PNP and GroEL(8, 18). A fairly detailed comparison between thedistances throughout the actin molecule when bound toprefoldin and TRiC/ADP could reveal if the stretching inprefoldin is the same as in TRiC/ADP, or if there isadditional effects on the actin molecule as it is beingtransferred from prefoldin to the TRiC cavity.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-13127 (URN)
    Tillgänglig från: 2008-04-03 Skapad: 2008-04-03
  • 264.
    Vorkapić, Emina
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Medicinska fakulteten.
    Targeting vascular remodeling in abdominal aortic aneurysm: To identify novel treatment strategies and drug candidates2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Abdominal aortic aneurysm (AAA) is a degenerative weakening of the aortic wall, mainly affecting elderly men with a prevalence of 4.4-7.7 %. AAA is characterized by medial and adventitial inflammatory cell infiltration associated with vascular remodeling of the extracellular matrix proteins such as collagen and elastin and with phenotypic modulation and loss of vascular smooth muscle cells (VSMCs). Although much research has been performed, the precise cellular and molecular pathways behind these processes are still poorly understood. The overall aim of this thesis was to target signaling pathways that affect vascular remodeling of AAA to potentially identify novel strategies and drug candidates for future treatment of aneurysmal diseases. In order to develop our understanding of the pathophysiology of AAA, we used the angiotensin (Ang) II-induced AAA animal model and human biopsies taken at end-stage of disease to recapitulate key aspects of disease formation.

    Innate immune receptors such as toll-like receptors (TLRs) are known to regulate immunological processes leading to the formation and progression of vascular disease including AAA. In paper I, we aimed to investigate the role of TLR signaling under the control of the TRIF adaptor protein in the formation of AAA. Human, aneurysmal aortas displayed increased expression of TLR3 and TLR4 in surface of macrophages and T lymphocytes. AngII-induced aneurysm formation was attenuated in mice lacking the Trif gene  (ApoE-/-Triʃ-/-), and these knockout mice presented with a more intact medial layer together with a reduced inflammatory response by macrophages and T lymphocytes and reduced levels of pro-inflammatory cytokines, chemokines, and proteases. Our results suggest an involvement of TRIF in the pathophysiology of AAA.

    Current management of AAA fully depends on imaging and surgical techniques, and drug-based therapies are still mostly ineffective. In paper II, we aimed to investigate the potential protective role of the tyrosine kinase inhibitor imatinib on the molecular mechanism involved in AAA formation. In AngII-infused ApoE-/-mice, 10 mg/kg imatinib per day affected several key features important in aneurysmal formation, including  preservation of the medial layer of the VSMCs, reduced infiltrations of CD3ε-positive T lymphocytes, and reduced gene expression of mast cell chymase, resulting in decreased aortic diameter and vessel wall thickness. These results highlight the importance of the tyrosine kinase inhibitor imatinib as a potential drug in the treatment of pathological vascular inflammation and remodeling in conditions such as AAA.

    In paper III, we aimed to investigate the role of adiponectin in experimentally induced AAA formation in mice. In mice with elevated adiponectin levels, AAA development was inhibited, and this was associated with reduced inflammatory cell infiltration, reduced medial degeneration of VSMCs and of elastin in the aortic vessel wall together with an improved systemic cytokine profile and the attenuation of periaortic adipose tissue (PVAT) inflammation. These results support the protective effect of adiponectin in the remodeling occurring in the aortic wall and in the prevention of AAA.

    In paper IV, we performed a descriptive study investigating the composition of PVAT adjacent to the aneurysmal aorta. We used immunohistochemistry to identify neutrophils, macrophages, mast cells, and T lymphocytes surrounding necrotic adipocytes in PVAT together with increased gene expression of IL-6 and cathepsin K and S. We also determined the concentrations of pro-inflammatory ceramides in PVAT and found an association to T lymphocytes. These results suggest that inflamed adipose tissue might be a source of proinflammatory cells and mediators that contribute to aortic wall degeneration.

    Delarbeten
    1. TRIF adaptor signaling is important in abdominal aortic aneurysm formation
    Öppna denna publikation i ny flik eller fönster >>TRIF adaptor signaling is important in abdominal aortic aneurysm formation
    Visa övriga...
    2015 (Engelska)Ingår i: Atherosclerosis, ISSN 0021-9150, E-ISSN 1879-1484, Vol. 241, nr 2, s. 561-568Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Objective: Abdominal aortic aneurysm (AAA) is characterized by inflammation, loss of smooth muscle cells (SMCs), and degradation of the extracellular matrix in the vessel wall. Innate immune receptors such as Toll-like receptors (TLRs) were recently shown to regulate immunological processes leading to the formation and progression of atherosclerotic plaques as well as to other cardiovascular pathologies. Our aim was to investigate whether blockage of TLR signaling, under the control of TIR domain-containing adaptor protein including IFN-beta (TRIF), could inhibit the inflammatory response and AAA development in mice. Results: In human AAA, an increased TLR3 and TLR4 expression in association with macrophages and T lymphocytes was demonstrated with immunohistochemical analysis. Angiotensin (Ang) II-induced aneurysm formation was significantly reduced by 30% in ApoE(-/-)Trif(-/-) mice compared to ApoE(-/-) mice. Morphologically, AngII-infused ApoE(-/-)Trif(-/-) mice had a more intact cellular and extracellular matrix while ApoE(-/-) mice infused with AngII displayed an increased medial thickness associated with aortic dissection, thrombus formation, and a more disorganized vessel wall. Gene expression analysis of the abdominal aorta revealed a profound decrease of the inflammatory genes CD68 (P less than 0.05), CD11b (P less than 0.05), and TNF-alpha (P less than 0.05) and the protease gene MMP-12 (P less than 0.01) in ApoE(-/-)Trif(-/-) mice compared to ApoE(-/-) mice infused with AngII. Conclusion: Our results suggest that signaling through TRIF is important for the inflammatory response of AngII-induced AAA and that blockage of the TRIF pathway reduces vascular inflammation and protects against AAA formation. (C) 2015 The Authors. Published by Elsevier Ireland Ltd.

    Ort, förlag, år, upplaga, sidor
    ELSEVIER IRELAND LTD, 2015
    Nyckelord
    Vascular disease; Inflammation; Toll-like receptor; Angiontensin II
    Nationell ämneskategori
    Klinisk medicin
    Identifikatorer
    urn:nbn:se:liu:diva-121438 (URN)10.1016/j.atherosclerosis.2015.06.014 (DOI)000360100700035 ()26100679 (PubMedID)
    Anmärkning

    Funding Agencies|Swedish Research Council [K2013-99X-22231-01-5]

    Tillgänglig från: 2015-09-18 Skapad: 2015-09-18 Senast uppdaterad: 2017-12-04
    2. Imatinib treatment attenuates growth and inflammation of angiotensin II induced abdominal aortic aneurysm
    Öppna denna publikation i ny flik eller fönster >>Imatinib treatment attenuates growth and inflammation of angiotensin II induced abdominal aortic aneurysm
    Visa övriga...
    2016 (Engelska)Ingår i: Atherosclerosis, ISSN 0021-9150, E-ISSN 1879-1484, Vol. 249, s. 101-109Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    AbstractBackground Abdominal aortic aneurysm (AAA) is characterized by vascular remodeling with increased infiltration of inflammatory cells and apoptosis/modulation of vascular smooth muscle cells (SMCs). Imatinib is a selective inhibitor of several tyrosine kinases, including PDGF receptors, Abl, and c-kit. The objective of this study was to characterize the potential protective role of imatinib on AAA development and the molecular mechanisms involved. Methods Male ApoE−/− mice were infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Daily treatment with 10 mg/kg imatinib, or tap water as control, was provided via gavage for 4 weeks. Results Treatment with imatinib was found to decrease the aortic diameter and vessel wall thickness, mediated by multiple effects. Imatinib treatment in AngII infused mice resulted in a reduced cellular infiltration of CD3ε positive T lymphocytes by 86% and reduced gene expression of mast cell chymase by 50% compared with AngII infused mice lacking imatinib. Gene expression analysis of SMC marker SM22α demonstrated an increase by 48% together with a more intact medial layer after treatment with imatinib as evaluated with SM22α immunostaining. Conclusion Present findings highlight the importance of tyrosine kinase pathways in the development of AAA. Our results show, that imatinib treatment inhibits essential mast cell, T lymphocyte and SMC mediated processes in experimental AAA. Thus, our results support the idea that tyrosine kinase inhibitors may be useful in the treatment of pathological vascular inflammation and remodeling in conditions like AAA.

    Nyckelord
    Abdominal aortic aneurysm, Vascular inflammation, Imatinib, Angiotensin II
    Nationell ämneskategori
    Cell- och molekylärbiologi Fysiologi
    Identifikatorer
    urn:nbn:se:liu:diva-127501 (URN)10.1016/j.atherosclerosis.2016.04.006 (DOI)000376505800016 ()27085160 (PubMedID)
    Tillgänglig från: 2016-04-28 Skapad: 2016-04-28 Senast uppdaterad: 2018-01-10Bibliografiskt granskad
    3. Inflammatory cells, ceramides, and expression of proteases in perivascular adipose tissue adjacent to human abdominal aortic aneurysms
    Öppna denna publikation i ny flik eller fönster >>Inflammatory cells, ceramides, and expression of proteases in perivascular adipose tissue adjacent to human abdominal aortic aneurysms
    Visa övriga...
    2017 (Engelska)Ingår i: Journal of Vascular Surgery, ISSN 0741-5214, E-ISSN 1097-6809, Vol. 65, nr 4, s. 1171-Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    BACKGROUND: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study.

    METHODS AND RESULTS: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT.

    CONCLUSIONS: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage.

    Nationell ämneskategori
    Kirurgi Kardiologi
    Identifikatorer
    urn:nbn:se:liu:diva-126051 (URN)10.1016/j.jvs.2015.12.056 (DOI)000402625400035 ()26960947 (PubMedID)
    Tillgänglig från: 2016-03-11 Skapad: 2016-03-11 Senast uppdaterad: 2018-05-07
  • 265.
    Wang, Hui
    et al.
    Wannan Med Coll, Peoples R China.
    Fang, Bin
    Anhui Univ, Peoples R China.
    Xiao, Lufei
    Chuzhou Vocat and Tech Coll, Peoples R China.
    Li, Di
    Wannan Med Coll, Peoples R China.
    Zhou, Le
    Wannan Med Coll, Peoples R China.
    Kong, Lin
    Anhui Univ, Peoples R China.
    Yu, Yan
    Wannan Med Coll, Peoples R China.
    Li, Xiangzi
    Wannan Med Coll, Peoples R China.
    Wu, Yunjun
    Wannan Med Coll, Peoples R China.
    Hu, Zhang-Jun
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär ytfysik och nanovetenskap. Linköpings universitet, Tekniska fakulteten.
    A water-soluble "turn-on" fluorescent probe for specifically imaging mitochondria viscosity in living cells2018Ingår i: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, E-ISSN 1873-3557, Vol. 203, s. 127-131Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Rational design of water-soluble probes for mitochondrial viscosity in practical biological applications remains a challenge. Herein, we described a novel hydro soluble benzothiazole salt derivative MitoSN, which exhibits specifically response and singular sensitivity to the mitochondria viscosity in living Hela cells. MitoSN displays an excellent fluorescence enhancement (ca. 35-fold) with the increase of the viscosity in the water-glycerol system. Moreover, confocal microscopy indicates that MitoSN is sensitive to the local viscosity and selectively stains mitochondria, the body of zebrafish as well. Importantly, MitoSN is capable to identify the viscosity difference of mitochondria in normal and nystatin treated Hela cells. The work provides a useful tool to monitor the changes of viscosity in the mitochondrial microenvironment. (C) 2018 Elsevier B.V. All rights reserved.

  • 266.
    Wang, Z. Q.
    et al.
    Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria; nternational Agency for Research on Cancer (IARC), F-69008 Lyon, France.
    Stingl, L.
    Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.
    Morrison, C.
    Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.
    Jantsch, M.
    Department of Cytology and Genetics, University of Vienna, A-1030 Vienna, Austria.
    Los, Marek Jan
    Department of Molecular Oncology/ Pediatry, German Cancer Research Center, D-69120 Heidelberg, Germany.
    Schulze-Osthoff, Klaus
    Medical Clinics, University of Tübingen, D-72076 Tübingen, Germany.
    Wagner, E. F.
    Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.
    PARP is important for genomic stability but dispensable in apoptosis1997Ingår i: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 11, nr 18, s. 2347-2358Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vive is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells Backing this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor alpha, gamma-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP-/-thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching and V(D)J recombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.

  • 267.
    Wassmer, Sarah
    et al.
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada.
    Rafat, Mehrdad
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, ON, Canada; University of Ottawa, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada.
    Fong, Wai Gin
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada.
    Baker, Adam N.
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada.
    Tsilfidis, Catherine
    Ottawa Hospital Research Institute, Vision Sciences Program, Ottawa, Canada; University of Ottawa, Department of Cellular and Molecular Medicine, Ottawa, ON, Canada; University of Ottawa, Department of Ophthalmology, Ottawa, ON, Canada.
    Chitosan microparticles for delivery of proteins to the retina2013Ingår i: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 9, nr 8, s. 7855-7864Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chitosan microparticles (CMPs) have previously been developed for topical applications to the eye, but their safety and efficacy in delivering proteins to the retina have not been adequately evaluated. This study examines the release kinetics of CMPs in vitro, and assesses their biocompatibility and cytotoxicity on retinal cells in vitro and in vivo. Two proteins were used in the encapsulation and release studies: BSA (bovine serum albumin) and tat-EGFP (enhanced green fluorescent protein fused to the transactivator of transcription peptide). Not surprisingly, the in vitro release kinetics were dependent on the protein encapsulated, with BSA showing higher release than tat-EGFP. CMPs containing encapsulated tat-EGFP were tested for cellular toxicity in photoreceptor-derived 661W cells. They showed no signs of in vitro cell toxicity at a low concentration (up to 1 mg ml 1), but at a higher concentration of 10 mg ml1 they were associated with cytotoxic effects. In vivo, CMPs injected into the subretinal space were found beneath the photoreceptor layer of the retina, and persisted for at least 8 weeks. Similar to the in vitro studies, the lower concentration of CMPs was generally well tolerated, but the higher concentration resulted in cytotoxic effects and in reduced retinal function, as assessed by electroretinogram amplitudes. Overall, this study suggests that CMPs are effective long-term delivery agents to the retina, but the concentration of chitosan may affect cytotoxicity.

  • 268.
    Wei, Yong
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska fakulteten. Univ Hlth Network, Canada; Struct Genom Consortium, Canada.
    Resetca, Diana
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Li, Zhe
    Yokohama City Univ, Japan.
    Johansson-Åkhe, Isak
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska fakulteten.
    Ahlner, Alexandra
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Helander, Sara
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för klinisk kemi. Linköpings universitet, Medicinska fakulteten.
    Wallenhammar, Amélie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Morad, Vivian
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Raught, Brian
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Wallner, Björn
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Bioinformatik. Linköpings universitet, Tekniska fakulteten.
    Kokubo, Tetsuro
    Yokohama City Univ, Japan.
    Tong, Yufeng
    Struct Genom Consortium, Canada; Univ Windsor, Canada.
    Penn, Linda Z.
    Univ Hlth Network, Canada; Univ Toronto, Canada.
    Sunnerhagen, Maria
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Multiple direct interactions of TBP with the MYC oncoprotein2019Ingår i: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 26, nr 11, s. 1035-+Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -angstrom resolution crystal structure reveals that human MYC amino acids 98-111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1(TAND1)). Using biochemical approaches, we have shown that MYC amino acids 115-124 also interact with TBP independently of TAF1(TAND1). Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.

  • 269.
    Wennerstrand, Patricia
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Blissing, Annica Theresia
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*82017Ingår i: ACS Omega, E-ISSN 2470-1343, Vol. 2, nr 8, s. 4991-4999Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

  • 270.
    Wesselborg, Sebastian
    et al.
    Department of Internal Medicine I, Eberhard-Karls-University, Tübingen, Germany. .
    Engels, I. H.
    Department of Internal Medicine I, Eberhard-Karls-University, Tübingen, Germany.
    Rossmann, E.
    Department of Internal Medicine I, Eberhard-Karls-University, Tübingen, Germany.
    Los, Marek Jan
    Department of Internal Medicine I, Eberhard-Karls-University, Tübingen, Germany.
    Schulze-Osthoff, Klaus
    Department of Internal Medicine I, Eberhard-Karls-University, Tübingen, Germany.
    Anticancer drugs induce caspase-8/FLICE activation and apoptosis in the absence of CD95 receptor/ligand interaction1999Ingår i: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 93, nr 9, s. 3053-3063Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling. (C) 1999 by The American Society of Hematology.

  • 271.
    Westendorp, Mo
    et al.
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Shatrov, Va
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Schulze-Osthoff, Klaus
    Division of Immunogenetics, German Cancer Research Center, Heidelberg. Germany .
    Frank, R.
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Kraft, M.
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Los, Marek Jan
    Division of Immunogenetics, German Cancer Research Center, Heidelberg, Germany.
    Krammer, Ph
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Droge, W.
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Lehmann, V.
    Division of Immunogenetics, German Cancer Research Center, Heidelberg..
    Hiv-1 Tat Potentiates Tnf-Induced Nf-Kappa-B Activation and Cytotoxicity1995Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 14, nr 3, s. 546-554Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    This study demonstrates that human immunodeficiency virus type 1 (HTV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HTV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein, TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat(1-72)), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio. They further imply a distinct mechanism of Mn-SOD suppression as compared,vith HIV-1 LTR transactivation by Tat. Taken together, our data suggest that Tat expressed in HIV-1-infected cells and Tat taken up by non-infected cells modulates TNF activity by altering the cellular redox state. These findings may be relevant for HIV-1 replication and for T cell depletion in acquired immune deficiency syndrome.

  • 272.
    Wu, Richard You
    et al.
    Hosp Sick Children, Canada; Univ Toronto, Canada.
    Li, Bo
    Hosp Sick Children, Canada.
    Koike, Yuhki
    Hosp Sick Children, Canada.
    Maattanen, Pekka
    Hosp Sick Children, Canada.
    Miyake, Hiromu
    Hosp Sick Children, Canada.
    Cadete, Marissa
    Hosp Sick Children, Canada.
    Johnson-Henry, Kathene C.
    Hosp Sick Children, Canada.
    Botts, Steven R.
    Hosp Sick Children, Canada.
    Lee, Carol
    Hosp Sick Children, Canada.
    Abrahamsson, Thomas
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för barns och kvinnors hälsa. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Barn- och kvinnocentrum, Barn- och ungdomskliniken i Linköping.
    Landberg, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk kemi.
    Pierro, Agostino
    Hosp Sick Children, Canada.
    Sherman, Philip M.
    Hosp Sick Children, Canada; Univ Toronto, Canada; Univ Toronto, Canada.
    Human Milk Oligosaccharides Increase Mucin Expression in Experimental Necrotizing Enterocolitis2019Ingår i: Molecular Nutrition & Food Research, ISSN 1613-4125, E-ISSN 1613-4133, Vol. 63, nr 3, artikel-id 1800658Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Scope Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. Methods and Results By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. Conclusions Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.

  • 273.
    Wäster, Petra
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Orfanidis, Kyriakos
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Hudkliniken i Östergötland.
    Eriksson, Ida
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Rosdahl, Inger
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Hjärt- och Medicincentrum, Hudkliniken i Östergötland.
    Seifert, Oliver
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Ryhov Hospital, Sweden.
    Öllinger, Karin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Region Östergötland, Diagnostikcentrum, Klinisk patologi och klinisk genetik.
    UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins-TGF-beta 1-FAP-alpha2017Ingår i: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 117, nr 4, s. 535-544Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-alpha is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-alpha is not yet completely revealed. Methods: Expression of FAP-alpha was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-beta 1. Results: Fibroblast activation protein-a expression was induced by UVR in melanocytes of human skin. The FAP-alpha expression was regulated by UVR-induced release of TGF-beta 1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-alpha mediated ECM degradation and facilitated tumour cell dissemination. Conclusions: Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-beta 1 and FAP-alpha expression, promoting cancer cell dissemination and melanoma metastatic spread.

  • 274.
    Wårdell, Karin
    Linköpings universitet, Institutionen för medicinsk teknik, Biomedicinsk instrumentteknik. Linköpings universitet, Tekniska fakulteten.
    Optical Monitoring Techniques for Navigation during Stereotactic Neurosurgery2016Ingår i: Sensors and materials, ISSN 0914-4935, Vol. 28, nr 10, s. 1105-1116Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Optical techniques are becoming increasingly important in healthcare, as both therapeutic and diagnostic tools. In neurosurgery, photonic technologies such as the microscope have already found widespread use. The overall aim of this paper is to give an overview of optical monitoring methods and their implementation and use in stereotactic and functional neurosurgery. The development steps presented in this paper range from radiofrequency-lesion size estimation using optics to a system adapted for neurosurgical intraoperative navigation during stereotactic deep brain stimulation (DBS) lead implantations. The two main optical methods implemented as intraoperative guidance tools are laser Doppler flowmetry and diffuse reflectance spectroscopy. By combining these fundamental methods with probe designs adapted to stereotactic systems, vessels can be tracked and "bar-codes" of tissue type along pre-defined trajectories used for DBS implantations identified. Examples of the optical methods used in relation to stereotactic DBS implantations are given.

  • 275.
    Yamashita, Tetsuji
    et al.
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Hakizimana, Pierre
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neuro- och inflammationsvetenskap. Linköpings universitet, Medicinska fakulteten. Karolinska Institutet, Stockholm, Sweden; Karolinska University Hospital, Stockholm, Sweden.
    Wu, Siva
    Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
    Hassan, Ahmed
    Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
    Jacob, Stefan
    Karolinska Institutet, Stockholm, Sweden.
    Temirov, Jamshid
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Fang, Jie
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Mellado-Lagarde, Marcia
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America; University of Brigthon, Brighton, United Kingdom.
    Gursky, Richard
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Horner, Linda
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Leibiger, Barbara
    Karolinska Institutet, Stockholm, Sweden.
    Leijon, Sara
    Karolinska Institutet, Stockholm, Sweden.
    Centonze, Victoria E
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Berggren, Per-Olof
    Karolinska Institutet, Stockholm, Sweden.
    Frase, Sharon
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Auer, Manfred
    Lawrence Berkeley National Laboratory, Berkeley, California,United States of America.
    Brownell, William E
    Baylor College of Medicine, Houston, Texas, United States of America.
    Fridberger, Anders
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten. Karolinska Institutet, Stockholm, Sweden; Karolinska University Hospital, Stockholm, Sweden.
    Zuo, Jian
    St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America.
    Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins2015Ingår i: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 11, nr 9, artikel-id e1005500Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  • 276.
    Yin, Lan
    et al.
    University of Gothenburg, Sweden.
    Vener Dödsbo, Alexander
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Spetea, Cornelia
    University of Gothenburg, Sweden.
    The membrane proteome of stroma thylakoids from Arabidopsis thaliana studied by successive in-solution and in-gel digestion2015Ingår i: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 154, nr 3, s. 433-446Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    From individual localization and large-scale proteomic studies, we know that stroma-exposed thylakoid membranes harbor part of the machinery performing the light-dependent photosynthetic reactions. The minor components of the stroma thylakoid proteome, regulating and maintaining the photosynthetic machinery, are in the process of being unraveled. In this study, we developed in-solution and in-gel proteolytic digestion methods, and used them to identify minor membrane proteins, e.g. transporters, in stroma thylakoids prepared from Arabidopsis thaliana (L.) Heynh Columbia-0 leaves. In-solution digestion with chymotrypsin yielded the largest number of peptides, but in combination with methanol extraction resulted in identification of the largest number of membrane proteins. Although less efficient in extracting peptides, in-gel digestion with trypsin and chymotrypsin led to identification of additional proteins. We identified a total of 58 proteins including 44 membrane proteins. Almost half are known thylakoid proteins with roles in photosynthetic light reactions, proteolysis and import. The other half, including many transporters, are not known as chloroplast proteins, because they have been either curated (manually assigned) to other cellular compartments or not curated at all at the plastid protein databases. Transporters include ATP-binding cassette (ABC) proteins, transporters for K+ and other cations. Other proteins either have a role in processes probably linked to photosynthesis, namely translation, metabolism, stress and signaling or are contaminants. Our results indicate that all these proteins are present in stroma thylakoids; however, individual studies are required to validate their location and putative roles. This study also provides strategies complementary to traditional methods for identification of membrane proteins from other cellular compartments.

  • 277.
    Zeng, Fan
    et al.
    Univ Innsbruck, Austria.
    Wunderer, Julia
    Univ Innsbruck, Austria.
    Salvenmoser, Willi
    Univ Innsbruck, Austria.
    Ederth, Thomas
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär fysik. Linköpings universitet, Tekniska fakulteten.
    Rothbaecher, Ute
    Univ Innsbruck, Austria.
    Identifying adhesive components in a model tunicate2019Ingår i: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 374, nr 1784, artikel-id 20190197Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tunicates populate a great variety of marine underwater substrates worldwide and represent a significant concern in marine shipping and aquaculture. Adhesives are secreted from the anterior papillae of their swimming larvae, which attach and metamorphose into permanently adhering, filter-feeding adults. We recently described the cellular composition of the sensory adhesive organ of the model tunicate Ciona intestinalis in great detail. Notably, the adhesive secretions of collocytes accumulate at the tip of the organ and contain glycoproteins. Here, we further explore the components of adhesive secretions and have screened for additional specificities that may influence adhesion or cohesion of the Ciona glue, including other carbohydrate moieties, catechols and substrate properties. We found a distinct set of sugar residues in the glue recognized by specific lectins with little overlap to other known marine adhesives. Surprisingly, we also detect catechol residues that likely originate from an adjacent cellular reservoir, the test cells. Furthermore, we provide information on substrate preferences where hydrophobicity outperforms charge in the attachment. Finally, we can influence the settlement process by the addition of hydrophilic heparin. The further analysis of tunicate adhesive strategies should provide a valuable knowledge source in designing physiological adhesives or green antifoulants. This article is part of the theme issue Transdisciplinary approaches to the study of adhesion and adhesives in biological systems.

  • 278.
    Ziels, Ryan
    et al.
    Linköpings universitet, Biogas Research Center. Civil and Environmental Engineering, University of Washington, WA, USA.
    Karlsson, Anna
    Linköpings universitet, Biogas Research Center. Scandinavian Biogas Fuels AB, Sweden.
    Beck, David A.C.
    Science Institute, University of Washington, WA, USA.
    Ejlertsson, Jörgen
    Linköpings universitet, Institutionen för tema, Tema Miljöförändring. Linköpings universitet, Biogas Research Center. Linköpings universitet, Filosofiska fakulteten. Scandinavian Biogas Fuels AB, Sweden.
    Shakeri Yekta, Sepehr
    Linköpings universitet, Institutionen för tema, Tema Miljöförändring. Linköpings universitet, Biogas Research Center. Linköpings universitet, Filosofiska fakulteten.
    Björn, Annika
    Linköpings universitet, Institutionen för tema, Tema Miljöförändring. Linköpings universitet, Biogas Research Center. Linköpings universitet, Filosofiska fakulteten.
    Stensel, H. David
    Civil and Environmental Engineering, University of Washington, WA, USA.
    Svensson, Bo H.
    Linköpings universitet, Institutionen för tema, Tema Miljöförändring. Linköpings universitet, Biogas Research Center. Linköpings universitet, Filosofiska fakulteten.
    Microbial community adaptation influences long-chain fatty acidconversion during anaerobic codigestion of fats, oils, and grease withmunicipal sludge2016Ingår i: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 103, s. 372-382Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Codigesting fats, oils, and greases with municipal wastewater sludge can greatly improve biomethanerecovery at wastewater treatment facilities. Process loading rates of fats, oils, and greases have beenpreviously tested with little knowledge of the digester microbial community structure, and high transientfat loadings have led to long chain fatty acid (LCFA) accumulation and digester upsets. This studyutilized recently-developed quantitative PCR assays for syntrophic LCFA-degrading bacteria along with16S amplicon sequencing to relate changes in microbial community structure to LCFA accumulationduring transient loading increases to an anaerobic codigester receiving waste restaurant oil andmunicipal wastewater sludge. The 16S rRNA gene concentration of the syntrophic b-oxidizing genusSyntrophomonas increased to ~15% of the Bacteria community in the codigester, but stayed below 3% inthe control digester that was fed only wastewater sludge. Methanosaeta and Methanospirillum were thedominant methanogenic genera enriched in the codigester, and together comprised over 80% of theArchaea community by the end of the experimental period. Constrained ordination showed that changesin the codigester Bacteria and Archaea community structures were related to measures of digester performance.Notably, the effluent LCFA concentration in the codigester was positively correlated to thespecific loading rate of waste oil normalized to the Syntrophomonas 16S rRNA concentration. Specificloading rates of 0e1.5 1012 g VS oil/16S gene copies-day resulted in LCFA concentrations below 30 mg/g TS, whereas LCFA accumulated up to 104 mg/g TS at higher transient loading rates. Based on thecommunity-dependent loading limitations found, enhanced biomethane production from high loadingsof fats, oils and greases can be achieved by promoting a higher biomass of slow-growing syntrophicconsortia, such as with longer digester solids retention times. This work also demonstrates the potentialfor controlling the loading rate of fats, oils, and greases based on the analysis of the codigester communitystructure, such as with quantitative PCR measurements of syntrophic LCFA-degrading bacteriaabundance.

  • 279.
    Zuse, Ann
    et al.
    Institute of Medicinal and Pharmaceutical Chemistry, Westphalian Wilhelms-University, Hittorfstrasse 58-62, D-48149 Münster, Germany; Manitoba Institute of Cell Biology, CancerCare Manitoba, Department of Biochemistry and Medical Genetics, Winnipeg, Canada.
    Prinz, Helge
    Institute of Medicinal and Pharmaceutical Chemistry, Westphalian Wilhelms-University, Hittorfstrasse 58-62, D-48149 Münster, Germany.
    Müller, Klaus
    Institute of Medicinal and Pharmaceutical Chemistry, Westphalian Wilhelms-University, Hittorfstrasse 58-62, D-48149 Münster, Germany.
    Schmidt, Peter
    Zentaris GmbH, Weismüllerstrasse 50, D-60314 Frankfurt, Germany.
    Günther, Eckhard G.
    Zentaris GmbH, Weismüllerstrasse 50, D-60314 Frankfurt, Germany.
    Schweizer, Frank
    Department of Chemistry, Univ. Manitoba, Winnipeg, Canada.
    Prehn, Jochen H.M.
    Department of Physiology and RCSI Research Institute, St. Stephen's Green, Dublin, Ireland.
    Los, Marek Jan
    Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    9-benzylidene-naphtho[2,3-b]thiophen-4-ones and benzylidene-9(10H)-anthracenones as novel tubulin interacting agents with high apoptosis-inducing activity2007Ingår i: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 575, nr 1-3, s. 34-45Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Tubulin-binding 9-benzylidene-naphtho[2,3-b]thiophen-4-ones 1a and 1b and benzylidene-9(10H)-anthracenone 2 were evaluated for their ability to induce cell death. We examined the effect of the molecules on cell cycle progression, organization of microtubule networks, and apoptosis induction. As determined by flow cytometry, cancer cells were predominantly arrested in metaphase with 4N DNA before cell death occurred. By using indirect immunofluorescence techniques we visualized microtubule depolymerization recognizable by short microtubule fragments scattered around the nucleus. The incubation with 1a and 2 resulted in chromatin condensation, nuclear fragmentation, and cell shrinkage, which are, among others, typical features of apoptotic cell death. Furthermore, time- and dose-dependent induction of apoptosis in SH-SY5Y cells was detected via cleavage of Ac-DEVD-AMC, a fluorigenic substrate for caspase-3. We observed a lower apoptotic activity in neuroblastoma cells overexpressing Bcl-xL, suggesting activation of the mitochondrial apoptosis pathway. Western blot analysis demonstrated that caspase-3, an apoptosis mediator, was activated in a time-dependent manner after exposure of SH-SY5Y cells to drugs 1a and 2. Taken together, the agents investigated in the present study display strong apoptosis-inducing activity and therefore show promise for the development of novel chemotherapeutics.

  • 280. Zuse, Anne
    et al.
    Prinz, Helge
    Mueller, Klaus
    Prehn, Jochen
    Los, Marek Jan
    BioApplications Enterprises, Winnipeg, MB, Canada; Manitoba Institute of Cell Biology, Cancer Care Manitoba; Manitoba Institute of Child Health; Department of Biochemistry and Medical Genetics; Department of Human Anatomy and Cell Science, University Manitoba, Winnipeg, Canada, .
    Design of novel small molecule inhibitors of tubulin polymerization with high apoptosis-inclucing activity2007Ingår i: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 6, nr 12, s. 3421S-3421SArtikel i tidskrift (Refereegranskat)
  • 281.
    Österberg, Emmy
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Teknisk biologi. Linköpings universitet, Tekniska högskolan.
    Ro52: Structure and interactions of constructs of RING and B-box2014Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    The ubiquitination process is vital to maintain the protein homeostasis in the cell. With high specificity it regulates degradation of proteins by tagging them with a small protein called ubiquitin. Four proteins are involved to perform the process and in this thesis one of these proteins is studied. This protein is called Ro52 and belongs to the TRIM protein family. It posses E3 ligase activity because of a N-terminal RING-domain and therefore it is responsible for the last step in the ubiquitination process. The structure of Ro52 is not totally solved and the function of the protein’s four domains is not fully understood.

    In this thesis three constructs of two domains from Ro52 (RING and B-box) is investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and auto-ubiquitination assay by Western blot. The goal was to gain deeper insight in structural and functional properties of these domains.

    In the end only two constructs were investigated because of time limitations. It was shown by NMR that one construct has similar structure as the wild type but lower stability, possibly due to shorter N-terminal region. Comparison of the results from CD measurements showed that the constructs were well structured but did not reveal any significant differences in secondary structure between the constructs. Functional analysis by Western blot encountered unexpected problems and no results were obtained.

    The current thesis provides a basis for further investigation of variant constructs jointly expressing the RING-B-box domains, and shows that even small changes may alter structure and stability in ways that might affect functional properties. 

3456 251 - 281 av 281
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