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  • 351. More, Jamileth
    et al.
    Galusso, Nadia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Veloso, Pablo
    Montecinos, Luis
    Pablo Finkelstein, José
    Sanchez, Gina
    Bull, Ricardo
    Luis Valdés, Jose
    Hidalgo, Cecilia
    Paula-Lima, Andrea
    N-Acetylcysteine Prevents the Spatial Memory Deficits and the Redox-Dependent RyR2 Decrease Displayed by an Alzheimer's Disease Rat Model2018Inngår i: Frontiers in Aging Neuroscience, ISSN 1663-4365, E-ISSN 1663-4365, Vol. 10, artikkel-id 399Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have previously reported that primary hippocampal neurons exposed to synaptotoxic amyloid beta oligomers (A beta Os), which are likely causative agents of Alzheimer's disease (AD), exhibit abnormal Ca2+ signals, mitochondrial dysfunction and defective structural plasticity. Additionally, A beta Os-exposed neurons exhibit a decrease in the protein content of type-2 ryanodine receptor (RyR2) Ca2+ channels, which exert critical roles in hippocampal synaptic plasticity and spatial memory processes. The antioxidant N-acetylcysteine (NAC) prevents these deleterious effects of A beta Os in vitro. The main contribution of the present work is to show that A beta Os injections directly into the hippocampus, by engaging oxidation-mediated reversible pathways significantly decreased RyR2 protein content but increased single RyR2 channel activation by Ca2+ and caused considerable spatial memory deficits. A beta Os injections into the CA3 hippocampal region impaired rat performance in the Oasis maze spatial memory task, decreased hippocampal glutathione levels and overall content of plasticity-related proteins (c-Fos, Arc, and RyR2) and increased ERK1/2 phosphorylation. In contrast, in hippocampus-derived mitochondria-associated membranes (MAM) A beta Os injections increased RyR2 levels. Rats fed with NAC for 3-weeks prior to A beta Os injections displayed comparable redox potential, RyR2 and Arc protein contents, similar ERK1/2 phosphorylation and RyR2 single channel activation by Ca2+ as saline-injected (control) rats. NAC-fed rats subsequently injected with A beta Os displayed the same behavior in the spatial memory task as control rats. Based on the present in vivo results, we propose that redox-sensitive neuronal RyR2 channels partake in the mechanism underlying A beta Os-induced memory disruption in rodents.

  • 352.
    Musdal, Yaman
    et al.
    Hacettepe University, Turkey.
    Ertan-Bolelli, Tugba
    Bolelli, Kayhan
    Yilmaz, Serap
    Ceyhan, Deniz
    Hegazy, Usama
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Aksoy, Yasemin
    Inhibition of human glutathione transferase P1-1 by novel benzazole derivatives2012Inngår i: Türk Biyokimya Dergisi, ISSN 0250-4685, E-ISSN 1303-829X, Vol. 37, nr 4, s. 431-436Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Objective: Glutathione transferases (GST) are multifunctional enzymes involved in detoxication, drug resistance, cell signaling and apoptosis. The inhibitory effects of novel benzazole derivatives were tested on human GST P1-1 to find new agents for overcoming drug resistance in cancer cells. Methods: GST P1-1 was heterogously expressed in E. coli strain XL-1 Blue and purified using S-hexylglutathione-Sepharose 6B affinity chromatography. The effect of 33 potential inhibitors on enzymatic activity was assayed spectrophotometrically with 1-chloro-2,4-dinitrobenzene (CDNB) as well as with the alternative substrate phenethyl isothiocyanate (PEITC). Results: Compound-18(N-[2-(4-chloro-benzyl)-benzooxazol-5-yl]-4-nitro-benzenesulfonamide) was the most potent inhibitor found with an IC50 value of approximately 10 mu M with respect to CDNB and a somewhat less strong inhibitor (45 % inhibition at 40 mu M) with PEITC as substrate. Compound-18 showed mixed inhibition with GSH and uncompetitive inhibition with CDNB with the K-i values 6.3 +/- 0.7 mu M and 11.8 +/- 3.4 mu M, respectively. Conclusion: Compound-18 is a potent inhibitor of GST P1-1. It may serve as a lead for further chemical modifications for increased potency. Additional studies will elucidate the effects of the inhibitor on cancer cells.

  • 353.
    Musdal, Yaman
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Govindarajan, Sridhar
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants2017Inngår i: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 30, nr 8, s. 543-549Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Exploring the vicinity around a locus of a protein in sequence space may identify homologs with enhanced properties, which could become valuable in biotechnical and other applications. A rational approach to this pursuit is the use of 'infologs', i.e. synthetic sequences with specific substitutions capturing maximal sequence information derived from the evolutionary history of the protein family. Ninety-five such infolog genes of poplar glutathione transferase were synthesized and expressed in Escherichia coli, and the catalytic activities of the proteins determined with alternative substrates. Sequence-activity relationships derived from the infologs were used to design a second set of 47 infologs in which 90% of the members exceeded wild-type properties. Two mutants, C2 (V55I/E95D/D108E/A160V) and G5 (F13L/C70A/G122E), were further functionally characterized. The activities of the infologs with the alternative substrates 1-chloro-2,4-dinitrobenzene and phenethyl isothiocyanate, subject to different chemistries, were positively correlated, indicating that the examined mutations were affecting the overall catalytic competence without major shift in substrate discrimination. By contrast, the enhanced protein expressivity observed in many of the mutants were not similarly correlated with the activities. In conclusion, small libraries of well-defined infologs can be used to systematically explore sequence space to optimize proteins in multidimensional functional space.

  • 354.
    Musdal, Yaman
    et al.
    Uppsala University, Sweden; Hacettepe University, Turkey.
    Hegazy, Usama M.
    Aksoy, Yasemin
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Uppsala University, Sweden.
    FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-12013Inngår i: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 205, nr 1, s. 53-62Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Objective: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. Methods: We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. Results: We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. Conclusion and practical implications: In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

  • 355.
    Musdal, Yaman
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Substrate specificities of two tau class glutathione transferases inducible by 2,4,6-trinitrotoluene in poplar2015Inngår i: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, nr 9, s. 1877-1883Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: The genome of poplar (Populus trichocarpa) encodes 81 glutathione transferases (GSTs) annotated in eight distinct classes. The tau class is considered the most versatile in the biotransformation of xenobiotics and is composed of 58 GSTs. Two of the enzymes, GSTU16 and GSTU45, have particular interest since their expression is induced by exposure of poplar tissues to 2,4,6-trinitrotoluene (TNT) and could potentially be involved in the metabolism of this toxic environmental contaminant.

    Results: DNA encoding these GSTs was synthesized and the proteins were heterologously expressed in Escherichia coli and the purified enzymes were characterized.

    Major conclusions: GSTU16 assayed with a number of conventional GST substrates showed the highest specific activity (60 mu mol min(-1) mg(-1)) with phenethyl isothiocyanate, 150-fold higher than that with CDNB. By contrast, GSTU45 showed CDNB as the most active substrate (3.3 mu mol min(-1) mg(-1)) whereas all of the 16 alternative substrates tested yielded significantly lower activities. Homology modeling suggested that the aromatic residues Phe10 and Tyr107 in the active site of GSTU16 are promoting the high activity with PEITC and other substrates with aromatic side-chains. Nonetheless, TNT was a poor substrate for GSTU16 as well as for GSTU45 with a specific activity of 0.05 nmol min(-1) mg(-1) for both enzymes. General significance: GSTU16 and GSTU45 do not play a major role in the degradation of TNT in poplar.

  • 356.
    Muñoz-Alarcón, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides and oligonucleotide delivery2012Licentiatavhandling, med artikler (Annet vitenskapelig)
  • 357.
    Muñoz-Alarcón, Andrés
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides and oligonucleotides: Design, uptake and therapeutic applications2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted.

    This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component.

  • 358.
    Muñoz-Alarcón, Andrés
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eriksson, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Novel efficient cell-penetrating peptide-mediated strategy for enhancing telomerase inhibitor oligonucleotidesManuskript (preprint) (Annet vitenskapelig)
  • 359.
    Muñoz-Alarcón, Andrés
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eriksson, Jonas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides2015Inngår i: Nucleic Acid Therapeutics, ISSN 2159-3337, E-ISSN 2159-3345, Vol. 25, nr 6, s. 306-310Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

  • 360.
    Muñoz-Alarcón, Andrés
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Guterstam, Peter
    Romero, Cristian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Behlke, Mark A.
    Lennox, Kim A.
    Wengel, Jesper
    EL Andaloussi, Samir
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Modulating Anti-MicroRNA-21 Activity and Specificity Using Oligonucleotide Derivatives and Length Optimization2012Inngår i: ISRN Pharmaceutics, ISSN 2090-6145, E-ISSN 2090-6153, Vol. 2012, artikkel-id 407154Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    MicroRNAs are short, endogenous RNAs that direct posttranscriptional regulation of gene expression vital for many developmental and cellular functions. Implicated in the pathogenesis of several human diseases, this group of RNAs provides interesting targets for therapeutic intervention. Anti-microRNA oligonucleotides constitute a class of synthetic antisense oligonucleotides used to interfere with microRNAs. In this study, we investigate the effects of chemical modifications and truncations on activity and specificity of anti-microRNA oligonucleotides targeting microRNA-21. We observed an increased activity but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination.

  • 361.
    Muñoz-Alarcón, Andrés
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Helmfors, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Webling, Kristin E. B.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptide fusion proteins2013Inngår i: Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges / [ed] Stefan R. Schmidt, Hoboken, N.J.: John Wiley & Sons, 2013, s. 397-411Kapittel i bok, del av antologi (Fagfellevurdert)
  • 362.
    Myrberg, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cell-penetrating peptides in cargo delivery: In vitro studies on uptake and in vivo studies on tumor targeting2008Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    The capability of cell-penetrating peptides (CPPs) to transport cargos over different cellular membranes both in vitro and in vivo have drawn major attention in the past decade. Three main topics on the application of CPPs have been studied in this thesis.

    First, several well-known CPPs, with fluorescein as a cargo, were shown to translocate into Nicotiana tabacum cultivar SR-1 protoplasts. By coupling different cargos to CPP it might be possible to effectively transport them inside the plant protoplasts. The translocation of CPPs into plant protoplasts might open up a new method for transformation of plant cells.

    Next, the cell-penetrating ability of the novel peptide YTA2 was characterized and it was established that chemical coupling between YTA2 and the protein cargo is not needed for the transport of the cargo over the cellular membrane in vitro. The delivery of proteins into cells by mere coincubation with CPPs is an improvement, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted.

    Finally, by conjugating each of the two breast tumor homing peptides, the cyclic cCPGPEGAGC (PEGA) or the linear CREKA peptide, to the CPP pVEC, two cell-penetrating peptides with homing properties were obtained. Both, PEGA-pVEC and CREKA-pVEC were taken up by different breast cancer cells in vitro. Moreover, the homing capacity of the PEGA-pVEC and CREKA-pVEC was conserved in vivo, where the conjugates mainly accumulated in blood vessels in breast tumor tissue and were subsequently translocated into cells. Conjugating the anti-cancer drug chlorambucil to PEGA-pVEC or CREKA-pVEC markedly improved its efficiency. Furthermore, systemic treatment of tumor-bearing mice with chlorambucil-CREKA-pVEC significantly reduced tumor growth compared to control groups. These tumor-homing CPPs might improve both diagnosis and treatment of breast cancer tumors, by conjugation to therapeutic agents.

  • 363.
    Myrberg, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindgren, Maria
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Protein delivery by the cell-penetrating peptide YTA22007Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 18, nr 1, s. 170-174Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In most cases, the transport of cell-penetrating peptide (CPP) with a cargo molecule over the plasma membrane requires a cross-linking of the cargo molecule to the peptide. Lately, a method of cargo delivery, coincubation with CPP, has been applied. We have studied uptake and toxicity of the CPP, YTA2, in the Bowes human melanoma cell line and human MDA-MB-231 breast cancer cell line and compared the results with known cell-penetrating peptides. The results show that fluoresceinyl YTA2 is taken up by the Bowes cells with 3.23 nmol/mg protein and shows low membrane toxicity to the cells with an EC50 of 60 μM. Furthermore, we show that YTA2 is capable of delivering cargo proteins, such as β-galactosidase and tetramethyl rhodamine iso-thiocyanate (TRITC) labeled streptavidin into cells by coincubation. The delivery of TRITC-labeled streptavidin was quantified to 42.4 pmol streptavidin/mg protein. The delivery of proteins into the cells by mere coincubation is an advantage, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted. In addition, the flexibility in CPP cargo delivery is increased.

  • 364.
    Myrberg, Helena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Zhang, Lianglin
    Mäe, Maarja
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Design of a Tumor-Homing Cell-Penetrating Peptide2008Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 19, nr 1, s. 70-75Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Chemotherapy is often limited by toxicity to normal cells. Therefore, an ideal anticancer drug should discriminate between normal tissue and tumors. This would require a target receptor molecule mostly present in tumors. The cyclic peptide cCPGPEGAGC (PEGA) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. PEGA peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pVEC, the conjugate is taken up by different breast cancer cells in vitro. Additionally, the homing capacity of the PEGA-pVEC is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. Furthermore, we show that the efficacy of the anticancer drug, chlorambucil, is increased more than 4 times when the drug is conjugated to the PEGA-pVEC chimeric peptide. These data demonstrate that combining a homing sequence with a cell-penetrating sequence yields a peptide that combines the desirable properties of the parent peptides. Such peptides may be useful in diagnostics and delivery of therapeutic agents to an intracellular location in a specific tumor target tissue.

  • 365.
    Mäe, Maarja
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Rational modifications of cell-penetrating peptides for drug delivery: Applications in tumor targeting and oligonucleotide delivery2009Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    High molecular weight biomolecules are becoming important in the development of new therapeutics. However, their size and nature creates a major limitation for their application – poor penetration through biological membranes. A new class of peptides, cell-penetrating peptides (CPPs), has shown the capability to transport various macromolecules inside the cells. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity and restricted bioavailability resulting from endocytic uptake of CPPs and entrapment in endosomal compartments.

    This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to achieve in vivo selectivity towards cancer cells. The first two papers employ tumor homing peptides as targeting moieties coupled to the N-termini of CPPs. In the third paper, a CPP is C-terminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific cleavage site followed by an inactivating amino acid sequence. In tissues overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated after proteolytic removal of the inactivating sequence, thus the cargo can be transported inside the cells. In paper IV, several CPPs have been N-terminally modified with a stearyl moiety and applied for the delivery of splice-correcting oligonucleotides. We show that stearyl-TP10 is as effective in oligonucleotide delivery as Lipofectamine™ 2000. Moreover, stearyl-TP10 has preserved efficacy in serum and is not toxic to cells. In conclusion, the rational modifications of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.

  • 366.
    Mäe, Maarja
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Karolinska Institutet, Sweden; University of Tartu, Estonia.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Lehto, Taavi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Chemically modified cell-penetrating peptides for the delivery of nucleic acids2009Inngår i: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 6, nr 11, s. 1195-1205Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Short nucleic acids targeting biologically important RNAs and plasmids have been shown to be promising future therapeutics; however, their hydrophilic nature greatly limits their utility in clinics and therefore efficient delivery vectors are greatly needed. Cell-penetrating peptides (CPPs) are relatively short amphipathic and/or cationic peptides that are able to transport various biologically active molecules inside mammalian cells, both in vitro and in vivo, in a seemingly non-toxic fashion. Although CPPs have proved to be appealing drug delivery vehicles, their major limitation in nucleic acid delivery is that most of the internalized peptide-cargo is entrapped in endosomal compartments following endocytosis and the bioavailability is therefore severely reduced. Several groups are working towards overcoming this obstacle and this review highlights the evidence that by introducing chemical modification in CPPs, the bioavailability of delivered nucleic acids increases significantly.

  • 367.
    Mäe, Maarja
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lundin, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Oskolkov, Nikita
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Johansson, Henrik J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Guterstam, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co-incubation strategy2009Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 134, nr 3, s. 221-227Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aberrations in splicing patterns play a significant role in several diseases, and splice correction, together with other forms of gene regulation, is consequently an emerging therapeutic target. In order to achieve successful oligonucleotide transfection, efficient delivery vectors are generally necessary. In this study we present one such vector, the chemically modified cell-penetrating peptide (CPP) TP10, for efficient delivery of a splice-correcting 2'-OMe RNA oligonucleotide. Utilizing a functional splice correction assay, we assessed the transfection efficiency of non-covalent complexes of oligonucleotides and stearylated or cysteamidated CPPs. Stearylation of the CPPs Arg9 and penetratin, as well as cysteamidation of MPG and TP10, did not improve transfection, whereas the presence of an N-terminal stearyl group on TP10 improved delivery efficiency remarkably compared to the unmodified peptide. The splice correction levels observed with stearyl-TP10 are in fact in parity with the effects seen with the commercially available transfection agent Lipofectamine (TM) 2000. However, the inherent toxicity associated with cationic lipid-based transfections can be completely eliminated when using the stearylated TP10, making this vector highly promising for non-covalent delivery of negatively charged oligonucleotides.

  • 368.
    Mäe, Maarja
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Enbäck, Juulia
    Laakkonen, Pirjo
    Rosenthal Aizman, Katri
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lindgren, Maria
    Target-activated cell-penetrating peptideManuskript (Annet vitenskapelig)
  • 369.
    Mäe, Maarja
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Myrberg, Helena
    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.
    Design of a tumor homing cell-penetrating peptide for drug delivery2009Inngår i: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 15, nr 1, s. 11-15Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.

  • 370. Mäe, Maarja
    et al.
    Zhang, Lianglin
    Myrberg, Helena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Ruoslahti, Erkki
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Inhibition of tumor growth by chlorambucil conjugated to a new tumor targeting peptide vectorManuskript (Annet vitenskapelig)
  • 371.
    Mäger, Imre
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Kent
    EL Andaloussi, Samir
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay2010Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1798, nr 3, s. 338-343Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.

  • 372. Nakase, Ikuhiko
    et al.
    Akita, Hidetaka
    Kogure, Kentaro
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Harashima, Hideyoshi
    Futaki, Shiroh
    Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides2012Inngår i: Accounts of Chemical Research, ISSN 0001-4842, E-ISSN 1520-4898, Vol. 45, nr 7, s. 1132-1139Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic add pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPS. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core shell nanoparticle with NAPS, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates programmed packaging to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.

  • 373.
    Nielsen, Henrietta M.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Mayo Clinic College of Medicine, USA.
    Chen, Kewei
    Lee, Wendy
    Chen, Yinghua
    Bauer, Robert J.
    Reiman, Eric
    Caselli, Richard
    Bu, Guojun
    Peripheral apoE isoform levels in cognitively normal APOE epsilon 3/epsilon 4 individuals are associated with regional gray matter volume and cerebral glucose metabolism2017Inngår i: Alzheimer's Research & Therapy, E-ISSN 1758-9193, Vol. 9, artikkel-id 5Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background

    Carriers of the APOE epsilon 4 allele are at increased risk of developing Alzheimer's disease (AD), and have been shown to have reduced cerebral metabolic rate of glucose (CMRgl) in the same brain areas frequently affected in AD. These individuals also exhibit reduced plasma levels of apolipoprotein E (apoE) attributed to a specific decrease in the apoE4 isoform as determined by quantification of individual apoE isoforms in APOE epsilon 4 heterozygotes. Whether low plasma apoE levels are associated with structural and functional brain measurements and cognitive performance remains to be investigated.

    Methods

    Using quantitative mass spectrometry we quantified the plasma levels of total apoE and the individual apoE3 and apoE4 isoforms in 128 cognitively normal APOE epsilon 3/epsilon 4 individuals included in the Arizona APOE cohort. All included individuals had undergone extensive neuropsychological testing and 25 had in addition undergone FDG-PET and MRI to determine CMRgl and regional gray matter volume (GMV).

    Results

    Our results demonstrated higher apoE4 levels in females versus males and an age-dependent increase in the apoE3 isoform levels in females only. Importantly, a higher relative ratio of apoE4 over apoE3 was associated with GMV loss in the right posterior cingulate and with reduced CMRgl bilaterally in the anterior cingulate and in the right hippocampal area. Additional exploratory analysis revealed several negative associations between total plasma apoE, individual apoE isoform levels, GMV and CMRgl predominantly in the frontal, occipital and temporal areas. Finally, our results indicated only weak associations between apoE plasma levels and cognitive performance which further appear to be affected by sex.

    Conclusions

    Our study proposes a sex-dependent and age-dependent variation in plasma apoE isoform levels and concludes that peripheral apoE levels are associated with GMV, CMRgl and possibly cognitive performance in cognitively healthy individuals with a genetic predisposition to AD.

  • 374. Nordin, Joel Z.
    et al.
    Lee, Yi
    Vader, Pieter
    Mäger, Imre
    Johansson, Henrik J.
    Heusermann, Wolf
    Wiklander, Oscar P. B.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Seow, Yiqi
    Bultema, Jarred J.
    Gilthorpe, Jonathan
    Davies, Tim
    Fairchild, Paul J.
    Gabrielsson, Susanne
    Meisner-Kober, Nicole C.
    Lehtio, Janne
    Smith, C. I. Edvard
    Wood, Matthew J. A.
    Andaloussi, Samir E. L.
    Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties2015Inngår i: Nanomedicine: Nanotechnology, Biology and Medicine, ISSN 1549-9634, E-ISSN 1549-9642, Vol. 11, nr 4, s. 879-883Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading toadifferent in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs.

  • 375.
    Nordin-Andersson, M
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Heldring, N
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dejongh, J
    Kjellstrand, P
    Walum, E
    Neurite degeneration in differentiated human neuroblastoma cells.1998Inngår i: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 12, nr 5, s. 557-60Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We have studied neurite degeneration in differentiated human neuroblastoma (SH-SY5Y) cells. The axonopathy-inducing potency in vitro of caffeine, diazepam, methylmercury chloride (MeHg), triethyltin chloride (TET) and acrylamide (ACR) was elucidated. After 72 hours of exposure the neurite degeneration was determined (by morphological quantification) as well as the total protein content (general cytotoxicity). The concentrations that caused 20% reduction of number of neurites (ND(20)) for ACR (250+/-36 mum) and TET (0.097+/-0.03 mum) was significantly lower, 63% and 35%, respectively (P</=0.005), as compared to corresponding inhibition of general cytotoxicity (IC(20)). The effects of TET on the neurites may be related to the disturbance in Ca(2+)-signalling, and thus a secondary event. The ND(20)s for caffeine and diazepam, which are compounds without a known neurite degenerative potency in vivo, were higher as compared with the IC(20). For MeHg which is an extremely cyto- and neurotoxic compound the ND(20) was not statistically different from the IC(20), indicating that degeneration of the neurites is not a primary effect. This study indicates that the SH-SY5Y-neurite degeneration model is useful for the identification of axonopathy-inducing substances.

  • 376.
    Nordin-Andersson, M
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Walum, E
    Kjellstrand, P
    Forsby, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery.2003Inngår i: Cell Biology and Toxicology, ISSN 0742-2091, E-ISSN 1573-6822, Vol. 19, nr 1, s. 43-51Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca2+]i) and receptor-activated (carbachol, 0.1 mmol/L) Ca2+ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca2+]i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca2+]i plays a significant role for acrylamide-induced axonopathy.

  • 377. Norrgard, Malena A.
    et al.
    Hellman, Ulf
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase2011Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, nr 19, s. 16871-16878Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.

  • 378. Norrgard, Malena A.
    et al.
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-Site Residue Sustaining Catalytic Promiscuity2011Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 412, nr 1, s. 111-120Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The substrate-binding H-site of human glutathione transferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 mu mol min(-1) mg(-1) resulted primarily from an increased k(cat) value (70 s(-1)). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 mu mol min(-1) mg(-1). In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conserved Ile10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrate selectivity profiles in GSTs by a few mutations.

  • 379. Ogaki, Kotaro
    et al.
    Martens, Yuka A.
    Heckman, Michael G.
    Koga, Shunsuke
    Labbe, Catherine
    Lorenzo-Betancor, Oswaldo
    Wernick, Anna I.
    Walton, Ronald L.
    Soto, Alexandra I.
    Vargas, Emily R.
    Nielsen, Henrietta M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Mayo Clinic, USA.
    Fujioka, Shinsuke
    Kanekiyo, Takahisa
    Uitti, Ryan J.
    van Gerpen, Jay A.
    Cheshire, William P.
    Wszolek, Zbigniew K.
    Low, Phillip A.
    Singer, Wolfgang
    Dickson, Dennis W.
    Bu, Guojun
    Ross, Owen A.
    Multiple system atrophy and apolipoprotein E2018Inngår i: Movement Disorders, ISSN 0885-3185, E-ISSN 1531-8257, Vol. 33, nr 4, s. 647-650Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Dysregulation of the specialized lipid metabolism involved in myelin synthesis and maintenance by oligodendrocytes has been associated with the unique neuropathology of MSA. We hypothesized that apolipoprotein E, which is associated with neurodegeneration, may also play a role in the pathogenesis of MSA. Objective: This study evaluated genetic associations of Apolipoprotein E alleles with risk of MSA and -synuclein pathology, and also examined whether apolipoprotein E isoforms differentially affect -synuclein uptake in a oligodendrocyte cell.

    Methods: One hundred sixty-eight pathologically confirmed MSA patients, 89 clinically diagnosed MSA patients, and 1,277 control subjects were genotyped for Apolipoprotein E. Human oligodendrocyte cell lines were incubated with -synuclein and recombinant human apolipoprotein E, with internalized -synuclein imaged by confocal microscopy and cells analyzed by flow cytometry.

    Results: No significant association with risk of MSA or was observed for either Apolipoprotein E 2 or 4. -Synuclein burden was also not associated with Apolipoprotein E alleles in the pathologically confirmed patients. Interestingly, in our cell assays, apolipoprotein E 4 significantly reduced -synuclein uptake in the oligodendrocytic cell line.

    Conclusions: Despite differential effects of apolipoprotein E isoforms on -synuclein uptake in a human oligodendrocytic cell, we did not observe a significant association at the Apolipoprotein E locus with risk of MSA or -synuclein pathology.

  • 380.
    Oglęcka, Kamila
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Magzoub, Mazin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Eriksson, L. E. Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Relevance of the N-terminal NLS-like sequence for membrane interactions of the Prion protein2007Inngår i: Biochimica et Biophysica Acta. MR. Reviews on Biomembranes, ISSN 0304-4157, E-ISSN 1879-257X, Vol. 1778, nr 1, s. 206-213Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23–28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1–28 (mPrPp(1–28)) and 23–50 (mPrPp(23–50)), respectively. In erythrocytes, mPrPp(1–28) induced 60% haemoglobin leakage after 30 min, whereas mPrPp(23–50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be 12% for 50 μM mPrPp(1–28), and 1% for 50 μM mPrPp(23–50). Circular dichroism spectra showed structure induction of mPrPp(1–28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23–50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1–28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23–50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.

  • 381.
    Oglęcka, Kamila
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Magzoub, Mazin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Eriksson, L. E. Göran
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects2008Inngår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, nr 1, s. 206-213Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23-28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1-28 (mPrPp(1-28)) and 2350 (rnPrPp(23-50)), respectively. In erythrocytes, mPrPp(1-28) induced similar to 60% haemoglobin leakage after 30 min, whereas mprPp(23-50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be similar to 12% for 50 mu M mPrPp(1-28), and similar to 1% for 50 mu M mPrPp(23-50). Circular dichroism spectra showed structure induction of mPrPp(1-28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mprPp(23-50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(I-28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23-50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.

  • 382. Oskolkov, Nikita
    et al.
    Arukuusk, Piret
    Copolovici, Dana-Maria
    Lindberg, Staffan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Margus, Helerin
    Padari, Kaert
    Pooga, Margus
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    NickFects, Phosphorylated Derivatives of Transportan 10 for Cellular Delivery of Oligonucleotides2011Inngår i: International journal of peptide research and therapeutics, ISSN 1573-3149, Vol. 17, nr 2, s. 147-157Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for nucleic acid polymers and, consequently, an efficient and non-toxic transfection agent is needed for their delivery into the cell. In this study we present a novel series, NickFects, of chemically modified TP10 peptide-based delivery vectors used for the cellular delivery of single-stranded oligonucleotides. These carriers, obtained by replacement of Ile8 by threonine in stearyl-TP10 and by modifying of tyrosine and/or threonine, respectively, by phosphorylation formed 300-500 nm in size peptide: oligonucleotide nanocomplexes with negative surface charges. The highest splice-correcting effect was obtained when phosphorotiate 2'-O-methyl oligonucleotides were transduced into cells by NickFect1 (NF1) or NickFect2 (NF2). In addition, we also show how a small modification (one or two negative charges) in peptide sequence can affect its ability to deliver ONs into cells and increase their potency in the splicing redirection assay. Our studies demonstrate that NF1 and NF2 have higher transfection efficacy for oligonucleotides as compared to the most commonly used transfection agent Lipofectamine (TM) 2000 and lead to higher biological response in cells.

  • 383. Padari, K.
    et al.
    Säälik, P.
    Hansen, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Koppel, K.
    Raid, R.
    Jiang, Y.
    Langel, Ü
    Pooga, M.
    Cell transduction pathways of transportans2005Inngår i: Bioconj. Chem., Vol. 16, s. 1399-1410Artikkel i tidsskrift (Fagfellevurdert)
  • 384. Padari, Kart
    et al.
    Koppel, Kaida
    Lorents, Annely
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mano, Miguel
    Pedroso de Lima, Maria C.
    Pooga, Margus
    S4(13)-PV Cell-Penetrating Peptide Forms Nanoparticle-Like Structures to Gain Entry Into Cells2010Inngår i: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 21, nr 4, s. 774-783Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.

  • 385. Pae, Janely
    et al.
    Liivamagi, Laura
    Lubenets, Dmitri
    Arukuusk, Piret
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pooga, Margus
    Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes2016Inngår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, nr 8, s. 1860-1867Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.

  • 386. Pae, Janely
    et al.
    Saeaelik, Pille
    Liivamaegi, Laura
    Lubenets, Dmitri
    Arukuusk, Piret
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Pooga, Margus
    Translocation of cell-penetrating peptides across the plasma membrane is controlled by cholesterol and microenvironment created by membranous proteins2014Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 192, s. 103-113Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Despite the extensive research in the field of CPPs' cell entry the exact mechanisms underlying their cellular uptake and the role of involved cell surface molecules in the internalization process have remained controversial. The present study focused on the interactions between CPPs and plasma membrane compounds using giant plasma membrane vesicles (GPMVs). GPMVs have shown to be a suitable model to study the translocation of CPPs across the plasma membrane in conditions lacking endocytosis. Our results show that higher cholesterol content and tighter packing of membrane predominantly reduce the accumulation of transportan, TP10 and model amphipathic peptide (MAP) in vesicles, indicating that the internalization of CPPs takes place preferentially via the more dynamic membrane regions. The partial digestion of membrane proteins from GPMVs' surface, on the other hand, drastically reduced the accumulation of nona-arginine and Tat peptide into vesicles, suggesting that proteins play a crucial role in the uptake of arginine-rich CPPs.

  • 387. Paern, Kalle
    et al.
    Viru, Liane
    Lehto, Taavi
    Oskolkov, Nikita
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Merits, Andres
    Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect62013Inngår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, nr 7Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

  • 388. Paernaste, Ly
    et al.
    Arukuusk, Piret
    Langel, Kent
    Tenson, Tanel
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    The Formation of Nanoparticles between Small Interfering RNA and Amphipathic Cell-Penetrating Peptides2017Inngår i: Molecular Therapy - Nucleic Acids, ISSN 2162-2531, E-ISSN 2162-2531, Vol. 7, s. 1-10Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are delivery vectors widely used to aid the transport of biologically active cargoes to intracellular targets. These cargoes include small interfering RNAs (siRNA) that are not naturally internalized by cells. Elucidating the complexities behind the formation of CPP and cargo complexes is crucial for understanding the processes related to their delivery. In this study, we used modified analogs of the CPP transportan10 and investigated the binding properties of these CPPs to siRNA, the formation parameters of the CPP/siRNA complexes, and their stabiliy to enzymatic degradation. We conclude that the pH dependent change of the net charge of the CPP may very well be the key factor leading to the high delivery efficiency and the optimal binding strength between CPPs to siRNAs, while the hydrophobicity, secondary structure of the CPP, and the positions of the positive charges are responsible for the stability of the CPP/siRNA particles. Also, CPPs with distinct hydrophobic and hydrophilic regions may assemble into nanoparticles that could be described as coreshell formulations.

  • 389.
    Palm Apergi, Caroline
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Design and evaluation of drug delivery vehicles2008Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    A crucial aspect of drug delivery is efficient transport to the site of action. Thus, there is a need to design and evaluate new delivery vehicles. In this thesis two delivery vehicles, cell-penetrating peptides and bacterial ghosts, were evaluated. The understanding of the internalization and degradation kinetics of cell-penetrating peptides is important for the practical aspects of cargo delivery since peptides have a notorious reputation of being rapidly degraded. If the cell-penetrating peptide remains intact inside the cellular environment, there is a possibility that the peptide-cargo conjugate leaks back to the extracellular environment. However, if it is degraded outside the cell, the cargo will never be delivered. In order to improve uptake efficiency and to be able to foresee side effects, the translocation mechanism needs to be fully elucidated. Data gathered from the first two papers led to the proposal of a new me-chanism involved in cell-penetrating peptide uptake: the membrane repair response, a resealing mechanism rapidly patching up broken membranes. This mechanism could explain the divergence in perception concerning the uptake pathways. Furthermore a new assay to produce the second delivery vehicle, bacterial ghosts, was developed based on data from the cell-penetrating peptide investigations. Bacterial ghosts are dead bacteria devoid of cytoplasmic contents but still retaining their structural and morphological characteristics, after protein E lysis of the bacterial cell membrane. By using a cell-penetrating peptide with antimicrobial effects, a new rapid peptide-based strategy to produce ghosts was developed and the capability to deliver plasmid DNA into the cell for expression was evaluated.

  • 390.
    Palm, Caroline
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Jayamanne, Mala
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Kjellander, Marcus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Peptide degradation is a critical determinant for cell-penetrating peptide uptake2007Inngår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1768, nr 7, s. 1769-1776Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.

  • 391.
    Palm-Apergi, Caroline
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    A new rapid cell-penetrating peptide based strategy to produce bacterial ghosts for plasmid delivery2008Inngår i: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 132, nr 1, s. 49-54Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The production of bacterial ghosts involves the lysis gene E plasmid in order to lyse and empty the bacteria of their cytoplasmic contents. After lysis the ghosts can either be loaded with new desired DNA and used for delivery to mammalian cells or used in vaccination. Cell-penetrating peptides have been used as delivery vehicles of drugs and oligonucleotides. Although many of them show low toxicity they have been compared to antimicrobial peptides involved in innate immunity. Recently we showed that cell-penetrating peptides also could be antimicrobial. In this study we take advantage of the antimicrobial effect of one cell-penetrating peptide, namely MAP, which is a model amphipathic peptide and treat bacteria with the peptide to produce bacterial ghosts. This new peptide based strategy is not dependent on the lysis gene E plasmid thus; several tiresome steps are removed in the production of ghosts. In addition the ghosts can be preloaded with a desired plasmid or DNA further removing time consuming reprocessing steps. To our knowledge this is the first study that uses a cell-penetrating peptide based strategy to produce bacterial ghosts to be used in plasmid delivery.

  • 392.
    Palm-Apergi, Caroline
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Lorents, Annely
    Padari, Kärt
    Pooga, Margus
    Hällbrink, Mattias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake2009Inngår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 23, nr 1, s. 214-223Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Although cell-penetrating peptides are able to deliver cargo into cells, their uptake mechanism is still not fully understood and needs to be elucidated to improve their delivery efficiency. Herein, we present evidence of a new mechanism involved in uptake, the membrane repair response. Recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. The direct penetration of hydrophilic peptides through the hydrophobic plasma membrane, however, is highly controversial. Three proteins involved in target cell apoptosis—perforin, granulysin, and granzymes—share many features common in uptake of cell-penetrating peptides (e.g., they bind proteoglycans). During perforin uptake, the protein activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, because of extracellular calcium influx. On activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane, resealing it within seconds. In this study, we have used flow cytometry, fluorescence, and electron microscopy, together with high-performance liquid chromatography and mass spectrometry, to present evidence that the membrane repair response is able to mask damages caused during cell-penetrating peptide uptake, thus preventing leakage of endogenous molecules out of the cell.—Palm-Apergi, C., Lorents, A., Padari, K., Pooga, M., and Hällbrink, M. The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake.

  • 393.
    Patra, Kalicharan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Soosaipillai, Antoninus
    Sando, Sigrid Botne
    Lauridsen, Camilla
    Berge, Guro
    Møller, Ina
    Grontvedt, Gøril Rolfseng
    Bråthen, Geir
    Begcevic, Ilijana
    Moussaud, Simon
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Minthon, Lennart
    Hansson, Oskar
    Diamandis, Eleftherios P.
    White, Linda R.
    Nielsen, Henrietta M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Assessment of kallikrein 6 as a cross-sectional and longitudinal biomarker for Alzheimer's disease2018Inngår i: Alzheimer's Research & Therapy, E-ISSN 1758-9193, Vol. 10, artikkel-id 9Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Kallikrein 6 (KLK6) is known to be an age-related protease expressed at high levels in the central nervous system. It was previously shown to be involved in proteolysis of extracellular proteins implicated in neurodegenerative diseases such as Alzheimer's disease (AD), prompting validation of KLK6 as a potential biomarker of disease. However, analyses of both plasma and cerebrospinal fluid (CSF) levels of KLK6 in patients with AD have been inconclusive. We present a detailed analysis of KLK6 in plasma and CSF in two separate cohorts in a cross-sectional and a longitudinal clinical setting. Methods: The cross-sectional cohort included control subjects without dementia and patients with AD, and the longitudinal cohort included patients with MCI and patients with AD followed over a 2-year period. Plasma and CSF levels of KLK6 were quantified by use of a previously developed and validated enzyme-linked immunosorbent assay. Statistical analyses were performed to compare KLK6 levels between diagnostic groups and to identify potential associations between KLK6 level, age, apolipoprotein E (APOE) genotype, total apoE level and the classical CSF AD biomarkers. Results: In the cross-sectional setting, KLK6 levels in plasma but not in CSF were significantly higher in the AD group than in control subjects. CSF but not plasma KLK6 levels were positively correlated with age in both the cross-sectional and longitudinal settings. In both cohorts, the CSF KLK6 levels were significantly and positively correlated with the CSF levels of core AD biomarkers. Total plasma and CSF apoE levels were positively associated with KLK6 in the cross-sectional study. Finally, during the 2-year monitoring period of the longitudinal cohort, CSF KLK6 levels increased with disease progression over time in the investigated patient groups. Conclusions: In two separate cohorts we have confirmed the previously reported correlation between age and CSF levels of KLK6. Increased plasma KLK6 levels in patients with AD with a more advanced disease stage suggest KLK6 as a potential biomarker in patients with AD with more severe dementia. Significant correlations between KLK6 levels and core CSF AD biomarkers suggest molecular links between KLK6 and AD-related pathological processes.

  • 394. Peritz, Tiina
    et al.
    Zeng, Fanyi
    Kannanayakal, Theresa J.
    Kilk, Kalle
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Eberwine, James
    Immunoprecipitation of mRNA-protein complexes2006Inngår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 1, nr 2, s. 577-580Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)–RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

  • 395.
    Pooga, Margus
    et al.
    Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Classes of Cell-Penetrating Peptides.2015Inngår i: Cell-Penetrating Peptides: Methods and Protocols, Springer-Verlag New York, 2015, Vol. 1324, s. 3-28Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    During the three decades of cell-penetrating peptides era the superfamily of CPPs has rapidly expanded, and the quest for new sequences continues. CPPs have been well recognized by scientific community and they have been used for transduction of a wide variety of molecules and particles into cultured cells and in vivo. In parallel with application of CPPs for delivering of active payloads, the mechanisms that such peptides take advantage of for gaining access to cells' insides have been in the focus of intense studies. Although the common denominator "cell penetration" unites all CPPs, the interaction partners on the cell surface, evoked cellular responses and even the uptake mechanisms might greatly vary between different peptide types. Here we present some possibilities for classification of CPPs based on their type of origin, physical-chemical properties, and the extent of modifications and design efforts. We also briefly analyze the internalization mechanisms with regard to their classification into groups based on physical-chemical characteristics.

  • 396. Pooga, Margus
    et al.
    Padari, Kärt
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Mechanisms of cargo delivery by transportans2007Inngår i: Handbook of cell-penetrating peptides / [ed] Ülo Langel, Boca Ranton: CRC Press, 2007, 2, s. 183-199Kapittel i bok, del av antologi (Fagfellevurdert)
  • 397. Prieto, Pilar
    et al.
    Blaauboer, Bas J
    de Boer, Albertus Gerrit
    Boveri, Monica
    Cecchelli, Romeo
    Clemedson, Cecilia
    Coecke, Sandra
    Forsby, Anna
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Galla, Hans-Joachim
    Garberg, Per
    Greenwood, John
    Price, Anna
    Tähti, Hanna
    Blood-brain barrier in vitro models and their application in toxicology. The report and recommendations of ECVAM Workshop 49.2004Inngår i: ATLA (Alternatives to Laboratory Animals), ISSN 0261-1929, Vol. 32, nr 1, s. 37-50Artikkel i tidsskrift (Fagfellevurdert)
  • 398. Pärn, Kalle
    et al.
    Eriste, Elo
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. Tartu University, Estonia.
    The Antimicrobial and Antiviral Applications of Cell-Penetrating Peptides2015Inngår i: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, Springer-Verlag New York, 2015, Vol. 1324, s. 223-245Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Over the past two decades, cell-penetrating peptides (CPPs) have become increasingly popular both in research and in application. There have been numerous studies on the physiochemical characteristics and behavior of CPPs in various environments; likewise, the mechanisms of entry and delivery capabilities of these peptides have also been extensively researched. Besides the fundamental issues, there is an enormous interest in the delivery capabilities of the peptides as the family of CPPs is a promising and mostly non-toxic delivery vector candidate for numerous medical applications such as gene silencing, transgene delivery, and splice correction. Lately, however, there has been an emerging field of study besides the high-profile gene therapy applications-the use of peptides and CPPs to combat various infections caused by harmful bacteria, fungi, and viruses.In this chapter, we aim to provide a short overview of the history and properties of CPPs which is followed by more thorough descriptions of antimicrobial and antiviral peptides. To achieve this, we analyze the origin of such peptides, give an overview of the mechanisms of action and discuss the various practical applications which are ongoing or have been suggested based on research.

  • 399. Pärnaste, Ly
    et al.
    Arukuusk, Piret
    Zagato, Elisa
    Braeckmans, Kevin
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Methods to follow intracellular trafficking of cell-penetrating peptides2016Inngår i: Journal of drug targeting (Print), ISSN 1061-186X, E-ISSN 1029-2330, Vol. 24, nr 6, s. 508-519Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems.

  • 400. Radwani, Houda
    et al.
    Lopez-Gonzalez, Maria José
    Cattaert, Daniel
    Roca-Lapirot, Olivier
    Dobremez, Eric
    Bouali-Benazzouz, Rabia
    Eiríksdóttir, Emelía
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Favereaux, Alexandre
    Errami, Mohammed
    Landry, Marc
    Fossat, Pascal
    Cav1.2 and Cav1.3 L-type calcium channels independently control short- and long-term sensitization to pain2016Inngår i: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 594, nr 22, s. 6607-6626Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    KEY POINTS: L-type calcium channels in the CNS exist as two subunit forming channels, Cav1.2 and Cav1.3, which are involved in short- and long-term plasticity. We demonstrate that Cav1.3 but not Cav1.2 is essential for wind-up. These results identify Cav1.3 as a key conductance responsible for short-term sensitization in physiological pain transmission. We confirm the role of Cav1.2 in a model of long-term plasticity associated with neuropathic pain. Up-regulation of Cav1.2 and down-regultation of Cav1.3 in neuropathic pain underlies the switch from physiology to pathology. Finally, the results of the present study reveal that therapeutic targeting molecular pathways involved in wind-up may be not relevant in the treatment of neuropathy.

    ABSTRACT: Short-term central sensitization to pain temporarily increases the responsiveness of nociceptive pathways after peripheral injury. In dorsal horn neurons (DHNs), short-term sensitization can be monitored through the study of wind-up. Wind-up, a progressive increase in DHNs response following repetitive peripheral stimulations, depends on the post-synaptic L-type calcium channels. In the dorsal horn of the spinal cord, two L-type calcium channels are present, Cav1.2 and Cav1.3, each displaying specific kinetics and spatial distribution. In the present study, we used a mathematical model of DHNs in which we integrated the specific patterns of expression of each Cav subunits. This mathematical approach reveals that Cav1.3 is necessary for the onset of wind-up, whereas Cav1.2 is not and that synaptically triggered wind-up requires NMDA receptor activation. We then switched to a biological preparation in which we knocked down Cav subunits and confirmed the prominent role of Cav1.3 in both naive and spinal nerve ligation model of neuropathy (SNL). Interestingly, although a clear mechanical allodynia dependent on Cav1.2 expression was observed after SNL, the amplitude of wind-up was decreased. These results were confirmed with our model when adapting Cav1.3 conductance to the changes observed after SNL. Finally, our mathematical approach predicts that, although wind-up amplitude is decreased in SNL, plateau potentials are not altered, suggesting that plateau and wind-up are not fully equivalent. Wind-up and long-term hyperexcitability of DHNs are differentially controlled by Cav1.2 and Cav1.3, therefore confirming that short- and long-term sensitization are two different phenomena triggered by distinct mechanisms.

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