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  • 301.
    Lindberg, Staffan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Muñoz-Alarcón, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tudoran, Oana
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mosqueira, Diogo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gyllborg, Daniel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    PepFect15, a novel endosomolytic cell-penetrating peptide for non-covalent oligonucleotide deliveryManuskript (preprint) (Annet vitenskapelig)
  • 302.
    Lindberg, Staffan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Regberg, Jakob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eriksson, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Muñoz-Alarcón, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Srimanee, Artita
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Figueroa, Ricardo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hallberg, Einar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ezzat, Kariem
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    A convergent uptake route for peptide- and polymer-based nucleotide delivery systems2015Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 206, s. 58-66Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

  • 303.
    Lindegren, H.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mogren, H.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi-Lilja, J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundqvist, J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Anionic linear aliphatic surfactants activate TRPV1: a possible endpoint for estimation of detergent induced eye nociception?2009Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 23, nr 8, s. 1472-1476Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The transient receptor potential vanilloid type 1 (TRPV1) has been reported as one of the key components in the pain pathway. Activation of the receptor causes a Ca2+ influx in sensory C-fibres with secondary effects leading to neurogenic inflammation in the surrounding tissue. We have earlier reported specific activation of TRPV1 by surfactant-containing hygiene products. We have continued this project by investigating activation of the TRPV1 by shampoo and soap ingredients in low concentrations measured as intracellular Ca2+ influxes in stably TRPV1-expressing neuroblastoma SH-SY5Y cells. As a TRPV1 specific control, the TRPV1 antagonist capsazepine was used. The response was quantified as the product induced Ca2+ influx during 2 min in relation to the maximum response induced by the TRPV1 agonist capsaicin. The results show that anionic alkyl linear surfactant ingredients such as sodium lauryl sulphate, sodium laureth sulphate, ammonium lauryl sulphate, sodium C12-15 pareth sulphate and N-lauroylsarcosine concentration-dependently induced Ca2+ influx that could be addressed to TRPV1. The cationic surfactants benzalkonium chloride and cetylpyridinium chloride induced a Ca2+ influx that was not TRPV1 mediated as well as the zwitterionic surfactant cocamidopropyl betaine, the non-linear anionic surfactant sodium deoxycholate and the non-ionic surfactant Triton-X. These results reveal a new mechanistic pathway for surfactant-induced nociception.

  • 304.
    Lindegren, Heléne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Alterations in receptor expression and function in scrapie-infected neuronal cell lines2004Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    This study shows that lipopolysaccharide (LPS) induces a robust, concentration-and time-dependent increase in nitric oxide (NO) production in the murine neuroblastoma cell line N2a by increased expression of iNOS. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was not due to inhibited enzymatic activity of iNOS but a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and reverse transcription-polymerase chain reaction (RT-PCR).

    Furthermore, major changes in the protein tyrosine phosphorylation profile of scrapie-infected hypothalamic neurons (ScGT1-1) and ScN2a, compared to their uninfected counterparts were shown, by Western blot of whole cell extracts and of anti-phosphotyrosine immunoprecipitates, with major bands corresponding to120, 150 and 180 kD. Anti-phosphotyrosine blots of lectin-purified glycoproteins, indicated that some, but not all, of the proteins in the 120 kD band are glycosylated. Two phosphoproteins of ≈ 120 kDa were identified, the receptor tyrosine kinase, fibroblast growth factor 2 (FGFR2) and the cytoplasmic non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (PYK2).

    In addition, scrapie-infection induces important alterations in the expression, binding and signalling of two neurotrophic receptors, the insulin-like growth factor-1 receptor (IGF-1R) and the insulin receptor (IR) in ScN2a, as compared to uninfected N2a cells.

    In ScN2a, the IGF-1R and IR protein levels were four- and two-fold increased, respectively, with an unexpected decrease in specific binding sites, as revealed by equilibrium binding studies. In the case of the IGF-1R, the apparent drop in binding sites was due to a seven-fold drop in IGF-1 affinity. Moreover, the IGF-1 stimulated IGF-1R tyrosine phosphorylation was increased in ScN2a when compared to the reduced affinity, possibly due to altered tyrosine kinase signalling in ScN2a. In the case of the IR, the binding affinity was unchanged, although insulin-stimulated IR tyrosine phosphorylation was increased.

  • 305.
    Lindegren, Heléne
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gyllberg, Hanna
    Östlund, Pernilla
    Bedecs, Katarina
    Altered protein tyrosine phosphorylation in scrapie-infected neuronal cell linesManuskript (Annet vitenskapelig)
  • 306.
    Lindegren, Heléne
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Östlund, Pernilla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gyllberg, Hanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bedecs, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Loss of lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase expression in scrapie-infected N2a cells2003Inngår i: Journal of neuroscience research, ISSN 0360-4012, Vol. 71, nr 2, s. 291-299Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In scrapie-infected cells, the conversion of the cellular prion protein to the pathogenic prion has been shown to occur in lipid rafts, which are suggested to function as signal transduction platforms. Neuronal cells may respond to bacterial lipopolysaccharide (LPS) treatment with a sustained and elevated nitric oxide (NO) release. Because prions and the major LPS receptor CD14 are colocalized in lipid rafts, the LPS-induced NO production in scrapie-infected neuroblastoma cells was studied. This study shows that LPS induces a dose- and time-dependent increase in NO release in the murine neuroblastoma cell line N2a, with a 50-fold increase in NO production at 1 g/ml LPS after 96 hr, as measured by nitrite in the medium. This massive NO release was not caused by activation of the neuronal NO synthase (nNOS), but by increased expression of the inducible NOS (iNOS) mRNA and protein. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was due not to abolished enzymatic activity of iNOS but to a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and RT-PCR. These results indicate that scrapie infection inhibits the LPS-mediated signal transduction upstream of the transcriptional step in the signaling cascade and may reflect the important molecular and cellular changes induced by scrapie infection

  • 307.
    Lindgren, Maria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    Classes and prediction of cell-penetrating peptides2011Inngår i: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2011, s. 3-19Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    The classical view on how peptides enter cells has been changed due to the development in the research field of cell-penetrating peptides (CPPs). During the last 15 years, more than 100 peptide sequences have been published to enter cells and also to bring different biological cargoes with them. Here, we present an overview of CPPs, mainly trying to analyze their common properties yielding the prediction of their cell-penetrating properties. Furthermore, examples of recent research, ideas on classification and uptake mechanisms, as well as a summary of the therapeutic potential of CPPs are presented.

  • 308.
    Lindgren, Maria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pooga, Margus
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Methods to study the translocation of cell-penetrating peptides2007Inngår i: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Raton: CRC Press, 2007, 2, s. 567-587Kapittel i bok, del av antologi (Fagfellevurdert)
  • 309.
    Lindgren, Maria
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rosenthal-Aizman, Katri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Saar, Külliki
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sassian, Meeri
    Östlund, Pernilla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide2006Inngår i: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 71, nr 4, s. 416-425Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide–MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 mM, the peptide–MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC50’s were determined for MTX, MTXYTA2 and YTA2 to be 18.5, 3.8 and 20 µM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells.

  • 310.
    Lindström, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Peer, Shawna M.
    Ing, Nancy H.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Characterization of equine GST A3-3 as a steroid isomerase2018Inngår i: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 178, s. 117-126Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ5-androstene-3,17-dione to Δ4-androstene-3,17-dione and of Δ5-pregnene-3,20-dione to Δ4-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals. The mRNA encoding the steroid isomerase GST A3-3 was cloned from testis of the horse (Equus ferus caballus). The concentrations of GSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. EcaGST A3-3 produced in E. coli has been characterized and shown to have highly efficient steroid double-bond isomerase activity, exceeding its activities with conventional GST substrates. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals and approaches the activity of the bacterial ketosteroid isomerase, one of the most efficient enzymes of all categories known today. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones.

  • 311. Lorents, Annely
    et al.
    Kodavali, Praveen Kumar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oskolkov, Nikita
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Pooga, Margus
    Cell-penetrating Peptides Split into Two Groups Based on Modulation of Intracellular Calcium Concentration2012Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 20, s. 16880-16889Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) promote the uptake of different cargo molecules, e.g. therapeutic compounds, making the harnessing of CPPs a promising strategy for drug design and delivery. However, the internalization mechanisms of CPPs are still under discussion, and it is not clear how cells compensate the disturbances induced by peptides in the plasma membrane. In this study, we demonstrate that the uptake of various CPPs enhances the intracellular Ca2+ levels in Jurkat and HeLa cells. The elevated Ca2+ concentration in turn triggers plasma membrane blebbing, lysosomal exocytosis, and membrane repair response. Our results indicate that CPPs split into two major classes: (i) amphipathic CPPs that modulate the plasma membrane integrity inducing influx of Ca2+ and activating downstream responses starting from low concentrations; (ii) non-amphipathic CPPs that do not evoke changes at relevant concentrations. Triggering of the membrane repair response may help cells to replace distorted plasma membrane regions and cells can recover from the influx of Ca2+ if its level is not drastically elevated.

  • 312. Lorents, Annely
    et al.
    Säälik, Pile
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pooga, Margus
    Arginine-Rich Cell-Penetrating Peptides Require Nucleolin and Cholesterol-Poor Subdomains for Translocation across Membranes2018Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 29, nr 4, s. 1168-1177Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesides (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nudeolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.

  • 313. Lundberg, P.
    et al.
    EL Andaloussi, S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sütlü, T.
    Johansson, H.
    Langel, Ü.
    Delivery of short interfering RNA using endosomolytic cell-penetrating peptides2007Inngår i: FASEB Journal, ISSN 0892-6638, Vol. 21, nr 11, s. 2664-2671Artikkel i tidsskrift (Fagfellevurdert)
  • 314.
    Lundberg, P
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kilk, K
    Langel, Ü
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptide mediated delivery of peptide nucleic acid oligomers2007Inngår i: Gene Transfer: Delivery and expression of DNA and RNA, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA , 2007, s. 581-586Kapittel i bok, del av antologi (Fagfellevurdert)
  • 315.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Naturally derived cell-penetrating peptides and applications in gene regulation: A study on internalization mechanisms and endosomal escape2006Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery.

    The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined.

    The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.

  • 316.
    Lundberg, Pontus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sütlü, Tolga
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Delivery of short interfering RNA using endosomolytic cell-penetrating peptides2007Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 21, nr 11, s. 2664-2671Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.—Lundberg, P., El-Andaloussi, S., Sütlü, T., Johansson, H., Langel, Ü. Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.

  • 317.
    Lundberg, Pontus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    El-Andaloussi, S
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sütlü, T
    Johansson, H
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, U
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Delivery of short interfering RNA using endosomolytic cell-penetrating peptides.2007Inngår i: FASEB J, ISSN 1530-6860, Vol. 21, nr 11, s. 2664-71Artikkel i tidsskrift (Fagfellevurdert)
  • 318.
    Lundberg, Pontus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Uptake mechanisms of novel cell-penetrating peptides derived from the Alzheimer’s disease associated gamma-secretase complex2006Inngår i: International journal of peptide research and therapeutics, ISSN 1573-3149, Vol. 12, nr 2, s. 105-114Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Basic peptides with vector abilities, so called cell-penetrating peptides (CPPs), have been reported to enter cells, carrying cargoes ranging from oligonucleotides and proteins to nanoparticles. In this study we present novel CPPs derived from the gamma-secretase complex, which is involved in the amyloidogenic processing of the amyloid precursor protein (APP) and one of the major research targets for Alzheimer’s disease therapeutics today. In order to examine the uptake efficiency and internalization mechanism of these novel CPPs, side-by-side comparison with the well characterized CPPs penetratin and tat were made. For assessment of the CPP uptake mechanism, endocytosis inhibitors, endosomal markers and cells deficient in the expression of glycosaminoglycans were used. Also, in order to determine the vector ability of the peptides, protein delivery was quantified.

    We demonstrate the uptake of the gamma-secretase derived CPPs, in accordance to penetratin and tat, to be largely dependent on temperature and initial binding to cell-surface glycosaminoglycans. After this initial step, there is a discrepancy in the mechanism of uptake, where all peptides, except one, is taken up by a PI 3-kinase dependent fluid phase endocytosis, which could be inhibited by wortmannin. Also, by using endosomal markers and protein delivery efficacy, we conclude that the pathway of internalization for different CPPs could determine the possible cargo size for which they can be used as a vector. The, in this study demonstrated, cell-penetrating properties of the gamma-secretase constituents could prove to be of importance for the gamma-secretase function, which is a matter of further investigation.

  • 319.
    Lundin, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides in delivery of splice-correcting oligonucleotides2008Licentiatavhandling, monografi (Annet vitenskapelig)
  • 320.
    Lundin, Per
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Toxicity methods for CPPs2011Inngår i: Cell-penetrating peptides: Methods and Protocols, New York: Humana Press, 2011, s. 195-205Kapittel i bok, del av antologi (Annet vitenskapelig)
    Abstract [en]

    CPPs have for numerous years been utilized as delivery vectors of various pharmaceutically interesting cargoes, both in vitro and in vivo. As CPPs are gradually approaching the bedsides, investigating toxicity associated with these highly interesting peptides becomes increasingly important and thorough initial assessment of cytotoxicity in vitro is a first step towards advancing these delivery vehicles in to the clinics. The present chapter describes protocols for four cytotoxicity assays in order to provide a toolbox for toxicity assessment of CPPs. The foci lie on membrane integrity (deoxyglucose leakage and propidium iodide assays) and cell viability (the MTT assay), but the chapter also provides a protocol for assessing an important parameter for future clinical applications, namely the hemolytic properties of CPPs.

  • 321.
    Lundin, Per
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Holm, Tina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hansen, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates2008Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, nr 12, s. 2535-2542Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are a growing family of peptides that have opened a new avenue in drug delivery, allowing various hydrophilic macromolecules to enter cells. In accordance with most other cationic delivery vectors, CPPs seem to rely mostly on endocytosis for internalization. However, due to conflicting results the exact endocytic pathways for CPP uptake have not yet been resolved. Here, we evaluated the ability of seven CPPs, with different chemical properties, to convey peptide nucleic acids (PNAs) inside cells. Assays based on both splice correction, generating biologically active read-out, and on traditional fluorescence measurements were utilized. The same assays were employed to assess different endocytic pathways and the dependence on extracellular heparan sulfates for internalization. Both highly cationic CPPs (M918, penetratin, and Tat) and amphipathic peptides (transportan, TP10, MAP, and pVEC) were investigated in this study. Conjugate uptake relied on endocytosis for all seven peptides but splice-correcting activity varied greatly for the investigated CPPs. The exact endocytic internalization routes were evaluated through the use of well-known endocytosis inhibitors and tracers. In summary, the different chemical properties of CPPs have little correlation with their ability to efficiently deliver splice-correcting PNA. However, conjugates of polycationic and amphipathic peptides appear to utilize different internalization routes.

  • 322.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Estimation of acute toxicity by using the differentiated neuronal progenitor C17.2 cell model2017Licentiatavhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The authorities in Europe and United States request information regarding possible toxicity for substances that are produced in one tonne or more per year. Estimation of acute systemic toxicity is conducted in vivo using mice or rats. These tests can be time consuming, costly, unethical, and in some cases irrelevant due to the lack of similarity between humans and rodents. It has been proposed that determining general cytotoxicity together with more tissue-specific effects assessed by using in vitro test systems, e.g. reflecting adverse structure or function in the nervous system, can be an alternative approach to the in vivo tests. Neurotoxicity studies in vitro can be performed by using primary cell cultures from fresh tissue or established cell lines, the latter being often preferred as they are beneficial both economically and ethically.

    Here, I present a murine neural progenitor cell line called C17.2 with the potential to differentiate to a mixed culture of both neurons and astrocytes. The differentiation process was examined using 3 different media compositions and 3 different exposures, totally 9 different scenarios. After 7 days in culture with DMEM/F-12 medium containing N2 supplements and 10 ng/mL nerve growth factor and 10 ng/mL brain derived neurotrophic factor, the culture contained two morphological distinguishable cell types, assumed to be neurons and astrocytes. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Western blot analyses were performed which confirmed the presence of neurons and astrocytes in the differentiated cultures. The mRNA and protein levels of the neuronal marker bIII-tubulin and the astrocyte marker glial fibrillary acidic protein (GFAP) were up-regulated during differentiation, while the progenitor cell marker nestin was down-regulated.

    To further investigate how this differentiated neural cell model responded to neurotoxic and non-neurotoxic substances, cell membrane potential (CMP) and mRNA expression of bIII-tubulin, GFAP and heat shock protein-32 were examined after exposure to nicotine, atropine, strychnine, ethanol, digoxin, and acetylsalicylic acid. The concentrations that induced effects on the CMP and biomarker expression were compared to general cytotoxicity (Inhibitory Concentration 50%) determined by the neutral red uptake assay in a mouse fibroblast 3T3 cell line, i.e. the 3T3/NRU assay. The CMP assay showed that nicotine, atropine and strychnine exposure induced depolarisation of the cell membrane. However, no effect on the CMP was seen when the cells were treated with acetylsalicylic acid, digoxin, and ethanol at non-cytotoxic concentrations. Alternation in the mRNA expression levels for one of the three biomarkers was seen at non-cytotoxic concentrations for all the compounds that induced acute toxicity by neuronal modes of action, i.e. nicotine, atropine and strychnine. No significant alteration was seen in any of the biomarker mRNA levels when the differentiated C17.2 cells were exposed to compounds that do not induce acute toxicity by neuronal modes of action, i.e. digoxin and acetylsalicylic acid and ethanol.

    In conclusion, acute toxicity, which could be induced by neuronal modes of action, may be detected in the differentiated C17.2 cell model by using CMP and gene expression biomarkers as endpoints. The simple cell culture requirements for culturing and differentiating the C17.2 cells into a mixed culture of neurons and astrocytes, the robustness in toxicity read-out, and the cost-effectiveness of the assay make the C17.2 cell line attractive as a model for acute neurotoxicity studies.

  • 323.
    Lundqvist, Jessica
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Neuroblastoma SH-SY5Y and neural progenitor C17.2 cell lines as models for neurotoxicological studies​2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    We are surrounded by chemicals, thus understanding how exposure to these chemicals affect us during our life is of great social importance. In order to predict human acute toxicity of chemicals, cosmetics or drugs, development of novel in vitro test strategies is required. The overall aim of this thesis was to evaluate whether two different cell line models could be used to predict acute neurotoxicity or developmental neurotoxicity. In paper one, we identified changes in cell membrane potential (CMP) as the most sensitive indicator of toxicity in neuroblastoma SH-SY5Y cells.

    In the following studies, we evaluated the capacity of the murine neural progenitor cell line C17.2 to differentiate into mixed cell cultures. Upon differentiation of the C17.2 cells we could identify two morphologically distinguishable cell types; astrocytes and neurons (Paper II). We then investigated how differentiated C17.2 cells responded to non-cytotoxic concentrations of three known neurotoxic and three non-neurotoxic substances. The neurotoxicants induced depolarisation of CMP and alteration in the mRNA expression of at least one of the three biomarkers studied, i.e. βIII-tubulin, glial fibrillary acidic protein or heat shock protein-32. In contrast, no significant effects were observed when exposed to non-neurotoxic compounds (Paper IV).

    To further characterise the C17.2 cell model during differentiation, an mRNA microarray analysis of the whole genome was performed. The 30 most significantly altered biomarkers with association to neuronal development were identified. The mRNA expression of the 30 biomarkers were used as a panel to alert for developmental neurotoxicity by exposing C17.2 cells during differentiation to toxicants known to induce impaired nervous system development. All but two of the selected genes were significantly altered by at least one of the chemicals, but none of the 30 genes were affected when treated with the negative control (Paper III).  

    In conclusion, the differentiated C17.2 neural progenitor cell line seems to be an attractive model for studying and predicting acute and developmental neurotoxicity. 

  • 324.
    Lundqvist, Jessica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Christina, Svensson
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kristina, Attoff
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Swetox, Karolinska Institutet, Sweden.
    Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity2017Inngår i: Applied In Vitro Toxicology, ISSN 2332-1539, Vol. 3, nr 2, s. 154-162Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Using general cytotoxicity assays in combination with in vitro tests for organ-specific toxicity has been proposed as an alternative approach to animal tests for estimation of acute systemic toxicity. Here, we present the C17.2 neural progenitor cell line as an option for estimation of acute neurotoxicity. The C17.2 cells were differentiated for 6 days in serum-free N2 medium with brain-derived neurotrophic factor and nerve growth factor to a mixed culture of neurons and astrocytes. The cells were then exposed to noncytotoxic concentrations of acetylsalicylic acid, atropine, digoxin, ethanol, nicotine, or strychnine for 48 hours and the mRNA levels of glial fibrillary acidic protein, βIII-tubulin, and heat shock protein 32 were analyzed as biomarkers for astrocytes, neurons, and cellular stress respectively. As a functional endpoint, the cell membrane potential (CMP) was monitored after acute addition of each compound to the differentiated C17.2 cells, by using the fluorescent FLIPR® membrane potential assay. Nicotine [3.2E-04 M], atropine [1.2E-05 M], or strychnine [6.4E-05 M] resulted in altered gene expression of at least one biomarker for each compound, indicating alerts for neurotoxicity. The three compounds also induced depolarization of the CMP at the lowest observed effect concentrations 9.5E-05 M of nicotine, 1.5E-05 M of atropine, and 6.9E-07 M of strychnine. The non-neurotoxic compounds acetylsalicylic acid, ethanol, and digoxin did neither affect the mRNA levels, nor the CMP. This study showed that the differentiated C17.2 cells might be useful for estimation of acute neurotoxicity by analyzing expression of mRNA biomarkers and CMP alterations.

  • 325.
    Lundqvist, Jessica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi-Lilja, Johanna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Svensson, Christina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gustafsson Dorfh, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests2013Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 27, nr 5, s. 1565-1569Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Here we present a multipotent neuronal progenitor cell line for toxicity testing as an alternative to primary cultures of mixed cell types from brain tissue. The v-myc immortalised C17.2 cell line, originally cloned from mouse cerebellar neural stem cells, were maintained as monolayer in cell culture dishes in DMEM supplemented with fetal calf serum, horse serum and antibiotics. Different media and exposure scenarios were used to induce differentiation. The optimal condition which generated mixed cultures of neurons and astrocytes included serum-free DMEM:F12 medium with N2 supplements, brain-derived neurotrophic factor and nerve growth factor. The medium was changed every 3rd or 4th day to fresh N2 medium with supplements. After 7days, the culture contained two different morphological cell types, assumed to be neurons and glia cells. The presence of astrocytes and neurons in the culture was confirmed by RT-PCR and Western blot analyses, indicating increased mRNA and protein levels of the specific biomarkers glial fibrillary acidic protein (GFAP) and βIII-tubulin, respectively. Concomitantly, the expression of the neural progenitor cell marker nestin was down-regulated.

  • 326.
    Lundström, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Subtype selective activation and molecular characterization of galanin receptors2007Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Showing an extensive distribution in the nervous system, and often in co-localization with the classical neurotransmitters, neuropeptides are functioning as important modulators of neuronal signaling. Subsequently, compelling evidence has implicated a modulatory role for the neuropeptide galanin in several physiological functions. The effect of galanin is trancduced intracellularly by three different receptors, and defining the explicit effect from these receptor subtypes is of outmost interest, and likely to result in future therapeutic utilization of the galanin system.

    The main aim of this thesis was to improve the development of subtype selective ligands utilized to differentiate between the galanin receptor subtypes. To achieve this, we have designed and developed novel galanin receptor ligands and characterized the molecular interactions necessary for ligand bindig at the GalR2 subtype.

    The major findings include the introduction and characterization of two galanin receptor ligands, selectively activating GalR1 or inhibiting GalR2. Although having moderate selectivity, the two ligands have been utilized in a number of studies, pursuing their initial presentation, in order to differentiate between the galanin receptors and to establish their specific function. Further optimization is likely to improve the selectivity and utilization of these ligands. By identifying the major pharmacophores in the Gal(2-11) ligand and the residues in the GalR2 subtype participating in ligand binding, we have been able to characterize the binding site in this receptor subtype and interactions that are of significance for recognition of subtype specific ligands. Together, these findings on GalR2 and Gal(2-11) are of importance for future design of ligands acting on this receptor.

  • 327.
    Lundström, Linda
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sollenberg, Ulla E.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Bartfai, Tamas
    Langel, Ulo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions2007Inngår i: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 103, nr 5, s. 1774-1784Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    To define the specific role of the galanin receptors when mediating the effect of galanin, effective tools for distinct activation and inhibition of the different receptor subtypes are required. Several of the physiological effects modulated by galanin are implicated to be mediated via the GalR2 subtype and have been distinguished from GalR1 effects by utilizing the Gal(2–11) peptide, recognizing only GalR2 and GalR3. In this study, we have performed a mutagenesis approach on the GalR2 subtype and present, for the first time, a molecular characterization of the interactions responsible for ligand binding and receptor activation at this receptor subtype. Our results identify four residues, His252 and His253 located in transmembrane domain 6 and Phe264 and Tyr271 in the extracellular loop 3, to be of great significance. We show evidence for the N-terminal tail of GalR2 to participate in ligand binding and that selective binding of Gal(2–11) includes interaction with the Ile256 residue, located at the very top of TM 6. In conclusion, we present a mutagenesis study on GalR2 and confer interactions responsible for ligand binding and receptor activation as well as selective recognition of the Gal(2–11) peptide at this receptor subtype. The presented observations could be of major importance for the design and development of new and improved peptide and non-peptide ligands, selectively activating the GalR2 subtype.

  • 328.
    Löfgren, Kajsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wahlström, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bedecs, Katarina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Antiprion properties of prion protein-derived cell-penetrating peptides2008Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, nr 7, s. 2177-2184Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In prion diseases, the cellular prion protein (PrPC) becomes misfolded into the pathogenic scrapie isoform (PrPSc) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1–1) and scrapie-infected (ScGT1–1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrPC or PrPSc. Treatment with the prion protein-derived CPPs mouse mPrP1–28 or bovine bPrP1–30 significantly reduced PrPSc levels in prion-infected cells but had no effect on PrPC levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1–1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP23–28, mPrP19–30, or mPrP23–50) had no effect on PrPSc levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrPSc and disables formation of prions.—Löfgren, K., Wahlström, A., Lundberg, P., Langel, U., Gräslund, A., and Bedecs, K. Antiprion properties of prion protein-derived cell-penetrating peptides.

  • 329.
    Löfgren Söderberg, Kajsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanisms of prion antagonization by PrP-derived cell-penetrating peptidesManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Cell penetrating peptides derived from the prion protein N-terminus (PrP-CPPs) reduce PrPSc levels in prion-infected neuronal cell cultures (1). The PrP-CPPs consist of the hydrophobic PrP signal sequence followed by a basic segment (KKRPKP) and enter cells through raft-dependent macropinocytosis. To decipher the PrP-CPP anti-prion mechanism, different peptide constructs were analyzed for effects on PrPSc levels in GT1-1 neuronal cell cultures infected with either prion strain RML or 22L. For both strains, the PrP-CPPs antagonized the infection, but RML and 22L-infections differed in sensitivity to the PrP-CPP anti-prion effect. We also show that the effect on PrPSc levels does not depend on peptide interaction with any chiral receptor. The signal sequence segment of the PrP-CPPs promotes a specific positioning within the cell where conversion may occur, as signal sequence segment shortening or targeting of the KKRPKP-motif into alternative sub-cellular compartments disrupts the peptide anti-prion effect. Defining the anti-prion mechanism of PrP-CPPs is a matter of establishing how the peptides connect to the prion replicative interface. As the conversion process is poorly understood, the PrP-CPPs represent useful tools to outline the sub-cellular context of prion propagation.

  • 330.
    Löfgren Söderberg, Kajsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Targeting prion propagation using peptide constructs with signal sequence motifs2014Inngår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 564, s. 254-261Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Synthetic peptides with sequences derived from the cellular prion protein (PrPc) unprocessed N-terminus are able to counteract the propagation of proteinase K resistant prions (PrPRes, indicating the presence of the prion isoform of the prion protein) in cell cultures (Lofgren et al., 2008). The anti-prion peptides have characteristics like cell penetrating peptides (CPPs) and consist of the prion protein hydrophobic signal sequence followed by a polycationic motif (residues KKRPKP), in mouse PrPc corresponding to residues 1-28. Here we analyze the sequence elements required for the anti-prion effect of KKRPKP-conjugates. Neuronal GT1-1 cells were infected with either prion strain RML or 22L Variable peptide constructs originating from the mPrP(1-28) sequence were analyzed for anti-prion effects, measured as disappearance of proteinase K resistant prions (PrPRes) in the infected cell cultures. We find that even a 5 amino acid N-terminal shortening of the signal peptide abolishes the anti-prion effect. We show that the signal peptide from PrPc can be replaced with the signal peptide from the Neural cell adhesion molecule-1; NCAMl(1-19), with a retained capacity to reduce PrPRes levels. The anti-prion effect is lost if the polycationic N-terminal PrPc-motif is conjugated to any conventional CPP, such as TAT(48-60), transportan-10 or penetratin. We propose a mechanism by which a signal peptide from a secretory or cell surface protein acts to promote the transport of a prion-binding polycationic PrPc-motif to a subcellular location where prion conversion occurs (most likely the Endosome Recycling Compartment), thereby targeting prion propagation.

  • 331.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Abdo, Rania
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hirose, Hisaaki
    Futaki, Shiroh
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient2013Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, nr 4, s. 1198-1204Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  • 332.
    Madani, Fatemeh
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Futaki, Shiroh
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanisms of Cellular Uptake of Cell-Penetrating Peptides2011Inngår i: Journal of Biophysics, ISSN 1687-8000, E-ISSN 1687-8019, artikkel-id 414729Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s).

  • 333.
    Mae, Maarja
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rautsi, Outi
    Enback, Juulia
    Hallbrink, Mattias
    Rosenthal Aizman, Katri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindgren, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Laakkonen, Pirjo
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Tumour targeting with rationally modified cell penetrating peptides2012Inngår i: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 18, nr 4, s. 361-371Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short transport peptides with a well-established ability for delivery of bioactive cargoes inside the cells both, in vitro and in vivo. CPPs enter unselectively in a wide variety of cell lines, this is a desirable property for most in vitro applications, however, in vivo e.g. in tumor models, specific targeted accumulation is required. In order to achieve tumor targeting, a known CPP, YTA4, was modified by prolonging it C-terminally with mainly negatively charged amino acids. Additionally, a matrix metalloproteinase-2 cleavage site was introduced between the CPP and the inactivating sequence. This new peptide, named NoPe, is an inactive pro-form of YTA4. It can be selectively cleaved and thereby activated by MMPs. We have conjugated an imaging agent, fluoresceinyl carboxylic acid, and a cytostatic agent methotrexate, to this activable pro-form. NoPe activation was demonstrated in vitro by recombinant MMP-2 cleavage and the cleavage of the attenuating sequence was abolished with MMP-2 specific inhibitor. Furthermore, the fluoresceinyl-NoPe is selectively accumulated in the tumor tissue in MDA-MB-231 tumor bearing mice after intravenous injection. Thus, this strategy proves to be successful for in vivo tumor imaging.

  • 334. Maeger, Imre
    et al.
    Langel, Kent
    Lehto, Taavi
    Eiriksdottir, Emelia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides2012Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, nr 3, s. 502-511Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.

  • 335.
    Magzoub, Mazin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Staffan, Sandgren
    Pontus, Lundberg
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oglecka, Kamila
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lilja, Johanna
    Wittrup, Anders
    Eriksson, L. E. Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Belting, Mattias
    Astrid, Gräslund
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis2006Inngår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 348, nr 2, s. 379-385Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1–24) and a basic domain (KKRPKP, residues 25–30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp–DNA–gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide’s ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases.

  • 336. Mann, Anita
    et al.
    Thakur, Garima
    Shukla, Vasundhara
    Singh, Anand Kamal
    Khanduri, Richa
    Naik, Rangeetha
    Jiang, Yang
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kalra, Namita
    Dwarakanath, B. S.
    Langel, Ulo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ganguli, Munia
    Differences in DNA Condensation and Release by Lysine and Arginine Homopeptides Govern Their DNA Delivery Efficiencies2011Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 8, nr 5, s. 1729-1741Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-Lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

  • 337.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Five Decades with Glutathione and the GSTome2012Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 9, s. 6072-6083Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept molecular quasi-species, we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution.

  • 338.
    Mannervik, Bengt
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sjödin, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Blood-Brain Barrier-Penetrating 6-Halogenopurines Suitable as Pro-Probes for Positron Emission Tomography are Substrates for Human Glutathione Transferases2016Inngår i: Pharmaceutical Bioprocessing, ISSN 2048-9145, Vol. 4, nr 2, s. 25-30Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    6-Chloro- and 6-bromopurines can cross the blood-brain barrier and in situ give rise to substrates of multidrug resistance-associated proteins (MRPs). The electrophilic purines form glutathione conjugates in reactions catalyzed by intracellular glutathione transferases (GSTs), and the conjugates are subsequently exported from the cells by ATP-dependent membrane transporters. In rodent model systems it has been demonstrated that suitably radiolabeled 6-halogenopurines by this scheme are pro-probes useful in monitoring the functionality of MRPs in intact brains using positron emission tomography. Prior to applications in human subjects it is imperative to establish the purine pro-probes as effective substrates for human GSTs occurring in brain and other tissues. We have developed a spectrophotometric assay for the glutathione conjugation and determined specific activities with a range of human GSTs as well as some rat GSTs for comparison. The ubiquitous GST P1-1 showed the highest activities with the 6-halogenopurines, which bodes well for the application of pro-probes for human investigations.

  • 339. Mansouri, Shiva
    et al.
    Barde, Swapnali
    Ortsäter, Henrik
    Eweida, Mohamed
    Darsalia, Vladimer
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Sjöholm, Åke
    Hökfelt, Tomas
    Patrone, Cesare
    GalR3 activation promotes adult neural stem cell survival in response to a diabetic milieu2013Inngår i: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 127, nr 2, s. 209-220Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type 2 diabetes impairs adult neurogenesis which could play a role in the CNS complications of this serious disease. The goal of this study was to determine the potential role of galanin in protecting adult neural stem cells (NSCs) from glucolipotoxicity and to analyze whether apoptosis and the unfolded protein response were involved in the galanin-mediated effect. We also studied the regulation of galanin and its receptor subtypes under diabetes in NSCs in vitro and in the subventricular zone (SVZ) in vivo. The viability of mouse SVZ-derived NSCs and the involvement of apoptosis (Bcl-2, cleaved caspase-3) and unfolded protein response [C/EBP homologous protein (CHOP) Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP), spliced X-box binding protein 1 (XBP1), c-Jun N-terminal kinases (JNK) phosphorylation] were assessed in the presence of glucolipotoxic conditions after 24h. The effect of diabetes on the regulation of galanin and its receptor subtypes was assessed on NSCs in vitro and in SVZ tissues isolated from normal and type 2 diabetes ob/ob mice. We show increased NSC viability following galanin receptor (GalR)3 activation. This protective effect correlated with decreased apoptosis and CHOP levels. We also report how galanin and its receptors are regulated by diabetes in vitro and in vivo. This study shows GalR3-mediated neuroprotection, supporting a potential future therapeutic development, based on GalR3 activation, for the treatment of brain disorders.

  • 340. Margus, Helerin
    et al.
    Arukuusk, Piret
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pooga, Margus
    Characteristics of Cell-Penetrating Peptide/Nucleic Acid Nanoparticles2016Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 13, nr 1, s. 172-179Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Nucleic acids are highly promising candidates for the treatment of various genetic diseases. However, due to the large size and negative charge, nucleic acids are not efficiently taken up by cells, and thus, their clinical potential remains limited so far. Therefore, various delivery vehicles have been designed to assist the cellular uptake of nucleic acids. Among these, cell-penetrating peptides (CPPs) have gained increasing popularity as efficient and nontoxic delivery vectors. CPPs can be coupled to nucleic acids either by covalent or noncovalent association. Noncovalent coupling, which is based on the formation of nanoparticle-like nanocomplexes (NP), has received much attention in recent years, and the number of studies employing the strategy is explosively increasing due to the high therapeutic potential. However, the properties of CPP/nucleic acid NPs have not been characterized in sufficient detail yet. We performed a comprehensive analysis of the size and morphology of nucleic acid nanoparticles with novel transfection peptides, PepFects (PFs) and NickFects (NFs), using negative staining transmission electron microscopy (TEM). In addition, we examined whether the attachment of fluorescence or (nano)gold label to nucleic acid affects the nanocomplex formation or its morphology. We demonstrated that transportan-10-based new generation CPPs from PF and NF families condense nucleic acids to NPs of homogeneous size and shape. The size and shape of assembled nanoparticles depend on the type of the complexed nucleic acid and the sequence of the used peptide, whereas the label on the nucleic acid does not influence the gross characteristics of formed NPs.

  • 341. Mattsson, Anna
    et al.
    Jeppsson, Fredrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. AstraZeneca CNS and Pain iMed, Södertälje, Sweden.
    Juréus, Anders
    Altered amyloid PET ligand binding to ApoE-containing plaques in Alzheimer´s disease brain in vitro Artikkel i tidsskrift (Fagfellevurdert)
  • 342.
    Mazari, Aslam M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dahlberg, Olle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Overexpression of Glutathione Transferase E7 in Drosophila Differentially Impacts Toxicity of Organic Isothiocyanates in Males and Females2014Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 10, artikkel-id e110103Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract. Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significantly increased survival when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies show a significantly lower survival rate. Oviposition activity was enhanced in transgenic flies. The effect was significant in transgenic females reared in the absence of ITCs as well as in the presence of 0.15 mM PEITC or 1 mM AITC. Thus the GSTE7 transgene elicits responses to exposure to ITC allelochemicals which differentially affect life-span and fecundity of male and female flies.

  • 343.
    Mazari, Aslam M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hegazy, Usama M.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Identification of new inhibitors for human hematopoietic prostaglandin D-2 synthase among FDA-approved drugs and other compounds2015Inngår i: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 229, s. 91-99Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Objective: Hematopoietic prostaglandin D-2 synthase (HPGDS) is a member of the Sigma class glutathione transferases (GSTs) catalyzing the isomerization of prostaglandin H-2 to prostaglandin D-2, a mediator of allergy and inflammation responses. Selective inhibitors of human HPGDS are expected to be of therapeutic importance in relieving symptoms related to allergy and asthma. Hence, a collection of diverse FDA-approved compounds was screened for potential novel applications as inhibitors of HPGDS. Methods: The catalytic activity of purified HPGDS was used for inhibition studies in vitro. Results: Our inhibition studies revealed 23 compounds as effective inhibitors of HPGDS with IC50 values in the low micromolar range. Erythrosine sodium, suramin, tannic acid and sanguinarine sulfate were characterized with IC50 values of 0.2, 0.3, 0.4, and 0.6 mu M, respectively. Kinetic inhibition analysis showed that erythrosine sodium is a nonlinear competitive inhibitor of HPGDS, while suramin, tannic acid and sanguinarine sulfate are linear competitive inhibitors. Conclusion: The results show that certain FDA-approved compounds may have pharmacological effects not previously realized that warrant further consideration in their clinical use.

  • 344.
    Mazari, Aslam M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene2016Inngår i: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 5, s. 141-145Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) and the related 2,4-dinitrotoluene (DNT) aretoxic environmental pollutants. The biotransformation and detoxication of these persistent compoundsin higher organisms are of great significance from a health perspective as well as for the biotechnological challenge of bioremediation of contaminated soil. We demonstrate that different human glutathionetransferases (GSTs) and GSTs from the fruit fly Drosophila melanogaster are catalysts of the biotransformationof TNT and DNT. The human GSTs had significant but modest catalytic activities with both DNT and TNT. However, D. melanogaster GSTE6 and GSTE7 displayed outstanding high activities withboth substrates.

  • 345.
    Mazari, Aslam M.A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Studies on Human and Drosophila melanogaster Glutathione Transferases of Biomedical and Biotechnological Interest2016Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Glutathione transferases (GSTs, EC.2.5.1.18) are multifunctional enzymes that are universally distributed in all cellular life forms. They play important roles in metabolism and detoxication of endogenously produced toxic compounds and xenobiotics. GSTs have gained considerable interest over the years for biomedical and biotechnological applications due to their involvement in the conjugation of glutathione (GSH) to a vast array of chemical species. Additionally, the emergence of non-detoxifying functions of GSTs has further increased their biological significance. The present work encompasses four scientific studies aimed at investigating human as well as fruit fly Drosophila melanogaster GSTs.

    Paper I presents the immobilization of GSTs on nanoporous alumina membranes. Kinetic analyses with 1-chloro-2,4-dinitrobenzene followed by specificity screening with alternative substrates showed a good correlation between the data obtained from immobilized enzymes and the enzymes in solution. Furthermore, immobilization showed no adverse effects on the stability of the enzymes. Paper II presents inhibition studies of human hematopoietic prostaglandin D2 synthase (HPGDS), a promising therapeutic target for anti-allergic and anti-inflammatory drugs. Our screening results with an FDA-approved drug library revealed a number of effective inhibitors of HPGDS with IC50 values in the low micromolar range. Paper III concerns the toxicity of organic isothiocyanates (ITCs) that showed high catalytic activities with GSTE7 in vitro. The in vivo results showed that phenethyl isothiocyanate (PEITC) and allyl isothiocyanate in millimolar dietary concentrations conferred toxicity to the adult fruit flies leading to death or shortened life-span. The transgenic female flies overexpressing GSTE7 showed increased tolerance against PEITC toxicity compared to the wild-type. However, the effect was opposite in male flies overexpressing GSTE7 after one week exposure. Notably, the transgene enhanced the oviposition activity of flies with and without ITCs exposure. Paper IV highlights Drosophila GSTs as efficient catalysts of the environmental pollutant and explosive 2,4,6-trinitrotoluene and the related 2,4-dinitrotoluene degradation. This result suggests the potential of GST transgenes in plants for biotransformation and phytoremediation of these persistent environmental pollutants. 

  • 346. Minami, S. Sakura
    et al.
    Min, Sang-Won
    Krabbe, Grietje
    Wang, Chao
    Zhou, Yungui
    Asgarov, Rustam
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Li, Yaqiao
    Martens, Lauren H.
    Elia, Lisa P.
    Ward, Michael E.
    Mucke, Lennart
    Farese, Robert V.
    Gan, Li
    Progranulin protects against amyloid beta deposition and toxicity in Alzheimer's disease mouse models2014Inngår i: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 20, nr 10, s. 1157-1164Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Haploinsufficiency of the progranulin (PGRN) gene (GRN) causes familial frontotemporal lobar degeneration (FTLD) and modulates an innate immune response in humans and in mouse models. GRN polymorphism may be linked to late-onset Alzheimer's disease (AD). However, the role of PGRN in AD pathogenesis is unknown. Here we show that PGRN inhibits amyloid beta (A beta) deposition. Selectively reducing microglial expression of PGRN in AD mouse models impaired phagocytosis, increased plaque load threefold and exacerbated cognitive deficits. Lentivirus-mediated PGRN overexpression lowered plaque load in AD mice with aggressive amyloid plaque pathology. A beta plaque load correlated negatively with levels of hippocampal PGRN, showing the dose-dependent inhibitory effects of PGRN on plaque deposition. PGRN also protected against A beta toxicity. Lentivirus-mediated PGRN overexpression prevented spatial memory deficits and hippocampal neuronal loss in AD mice. The protective effects of PGRN against A beta deposition and toxicity have important therapeutic implications. We propose enhancing PGRN as a potential treatment for PGRN-deficient FTLD and AD.

  • 347. Minami, S. Sakura
    et al.
    Shen, Vivian
    Le, David
    Krabbe, Grietje
    Asgarov, Rustam
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Gladstone Institute of Neurological Diseases, United States.
    Perez-Celajes, Liberty
    Lee, Chih-Hung
    Li, Jinhe
    Donnelly-Roberts, Diana
    Gan, Li
    Reducing inflammation and rescuing FTD-related behavioral deficits in progranulin-deficient mice with alpha 7 nicotinic acetylcholine receptor agonists2015Inngår i: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 97, nr 4, s. 454-462Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Mutations in the progranulin gene cause frontotemporal dementia (FTD), a debilitating neurodegenerative disease that involves atrophy of the frontal and temporal lobes and affects personality, behavior, and language. Progranulin-deficient mouse models of FTD exhibit deficits in compulsive and social behaviors reminiscent of patients with FTD, and develop excessive microgliosis and increased release of inflammatory cytokines. Activation of nicotinic acetylcholine receptors (nAChRs) by nicotine or specific alpha 7 nAChR agonists reduces neuroinflammation. Here, we investigated whether activation of nAChRs by nicotine or alpha 7 agonists improved the excessive inflammatory and behavioral phenotypes of a progranulin-deficient FTD mouse model. We found that treatment with selective alpha 7 agonists, PHA-568487 or ABT-107, strongly suppressed the activation of NF-kappa B in progranulin-deficient cells. Treatment with ABT-107 also reduced microgliosis, decreased TNF alpha levels, and reduced compulsive behavior in progranulin-deficient mice. Collectively, these data suggest that targeting activation of the alpha 7 nAChR pathway may be beneficial in decreasing neuroinflammation and reversing some of the behavioral deficits observed in progranulin-deficient FTD.

  • 348. Mitsueda, Asako
    et al.
    Shimatani, Yuri
    Ito, Masahiro
    Ohgita, Takashi
    Yamada, Asako
    Hama, Susumu
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Harashima, Hideyoshi
    Nakase, Ikuhiko
    Futaki, Shiroh
    Kogure, Kentaro
    Development of a Novel Nanoparticle by Dual Modification With the Pluripotential Cell-Penetrating Peptide PepFect6 for Cellular Uptake, Endosomal Escape, and Decondensation of an siRNA Core Complex2013Inngår i: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 100, nr 6, s. 698-704Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 698-704, 2013.

  • 349. Moden, Olof
    et al.
    Zhang, Wei
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine2012Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 25, nr 4, s. 189-197Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structureactivity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with approximate to 10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93 library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.

  • 350. Modén, Olof
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    Glutathione Transferases in the Bioactivation of Azathioprine2014Inngår i: Redox and Cancer Part A, San Diego: Elsevier, 2014, s. 199-244Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adverse side effects. The prodrug is activated by glutathione conjugation and release of 6-mercaptopurine, a reaction most efficiently catalyzed by glutathione transferase (GST) A2-2. Among five genotypes of GST A2-2, the variant A2*E has threefold fourfold higher catalytic efficiency with azathioprine, suggesting that the expression of A2*E could boost 6-mercaptopurine release and adverse side effects in treated patients. Structure-activity studies of the GST A2-2 variants and homologous alpha class GSTs were made to delineate the determinants of high catalytic efficiency compared to other alpha class GSTs. Engineered chimeras identified GST peptide segments of importance, and replacing the corresponding regions in low-activity GSTs by these short segments produced chimeras with higher azathioprine activity. By contrast, H-site mutagenesis led to decreased azathioprine activity when active-site positions 208 and 213 in these favored segments were mutagenized. Alternative substitutions indicated that hydrophobic residues were favored. A pertinent question is whether variant A2*E represents the highest azathioprine activity achievable within the GST structural framework. This issue was addressed by mutagenesis of H-site residues assumed to interact with the substrate based on molecular modeling. The mutants with notably enhanced activities had small or polar residues in the mutated positions. The most active mutant L107G/L108D/F222H displayed a 70-fold enhanced catalytic efficiency with azathioprine. The determination of its structure by X-ray crystallography showed an expanded H-site, suggesting improved accommodation of the transition state for catalysis.

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