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  • 1. Almuzzaini, Bader
    et al.
    Sarshad, Aishe A.
    Rahmanto, Aldwin S.
    Hansson, Magnus L.
    Von Euler, Anne
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sangfelt, Olle
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Percipalle, Piergiorgio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    In beta-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects2016In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 30, no 8, 2860-2873 p.Article in journal (Refereed)
    Abstract [en]

    Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that beta-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of beta-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In beta-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type beta-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of beta-actin in Pol I transcription. The rRNA synthesis defects in the beta-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (mono-methylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated beta-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.

  • 2.
    Cerrato, Carmine Pasquale
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pirisinu, Marco
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Sassari, Italy.
    Vlachos, Efstathios Nikolaos
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Novel cell-penetrating peptide targeting mitochondria2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, no 11, 4589-4599 p.Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short, nontoxic peptides with cationic and/or amphipathic properties able to cross the cellular membrane. CPPs are used for the delivery of a wide variety of cargoes, such as proteins, oligonucleotides, and therapeutic molecules. The aim of the present study was to synthesize unusually small novel CPPs targeting mitochondria based on the Szeto-Schiller peptide (SS-31) to influence intramitochondrial processes and to improve the biologic effects. All the peptides used were synthesized manually using 9-fluorenylmethyloxycarbonyl chemistry. In the first part of the study, HeLa 705, U87, and bEnd.3 cells were used as in vitro delivery model. Cells were incubated for 24 h at 37°C and 5% CO2 with different concentrations of our peptides. Cell proliferation assay was performed to evaluate cell viability. Biologic effects such as mitochondrial membrane potential and antioxidant activity were evaluated. H2O2 was used as positive control. Uptake studies were performed using peptides conjugated with 5(6)-carboxyfluorescein (FAM). Fluorescent microscopy was used to determine presence and localization of peptides into the cells. Isolated mitochondria from pretreated cells and mitochondria treated after isolation were used to confirm the targeting ability of the peptide. Uptake of FAM alone was used as negative control. Microscopy studies confirmed the ability of peptides to penetrate cell. Localization analysis showed increase in uptake by 35% compared with SS-31. Mitochondrial CPP 1 (mtCPP-1) had no effect on mitochondrial membrane potential and prevented reactive oxygen species formation in bEnd.3 cells by 2-fold compared with SS-31. No cytotoxicity was observed even at high concentration (100 µM). These data suggest that mtCPP-1 is a mitochondrial CPP and protect mitochondria from oxidative damage due to its own antioxidant activities.-Cerrato, C. P., Pirisinu M., Vlachos E. N., Langel, Ü. Novel cell-penetrating peptide targeting mitochondria.

  • 3.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Helmfors, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Tudoran, Oana
    Juks, Carmen
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Padari, Kärt
    EL Andaloussi, Samir
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Scavenger receptor-mediated uptake of cell-penetrating peptide nanoparticles with oligonucleotides2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 3, 1172-1180 p.Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are shortcationic peptides that penetrate cells by interacting withthe negatively charged plasma membrane; however, thedetailed uptake mechanism is not clear. In contrary to theconventional mode of action of CPPs, we show here thata CPP, PepFect14 (PF14), forms negatively charged nanocomplexeswith oligonucleotides and their uptake is mediatedby class-A scavenger receptors (SCARAs). Specificinhibitory ligands of SCARAs, such as fucoidin, polyinosinicacid, and dextran sulfate, totally inhibit the activityof PF14-oligonucleotide nanocomplexes in the HeLapLuc705 splice-correction cell model, while nonspecific,chemically related molecules do not. Furthermore, RNAinterference (RNAi) knockdown of SCARA subtypes(SCARA3 and SCARA5) that are expressed in this cell lineled to a significant reduction of the activity to <50%. Inline with this, immunostaining shows prevalent colocalizationof the nanocomplexes with the receptors, and electronmicroscopy images show no binding or internalizationof the nanocomplexes in the presence of theinhibitory ligands. Interestingly, naked oligonucleotidesalso colocalize with SCARAs when used at high concentrations.These results demonstrate the involvement ofSCARA3 and SCARA5 in the uptake of PF14-oligonucleotidenanocomplexes and suggest for the first time thatsome CPP-based systems function through scavenger receptors,which could yield novel possibilities to understandand improve the transfection by CPPs.

  • 4. Franks, P. W.
    et al.
    Scheele, C
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Loos, R. J. F.
    Nielsen, A. R.
    Finucane, F. M.
    Wahlestedt, C.
    Pedersen, B. K.
    Wareham, N. J.
    Timmons, J. A.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Genomic variants at the PINK1 locus are associated with transcript abundance and plasma nonesterified fatty acid concentrations in European whites2008In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, no 9, 3135-3145 p.Article in journal (Refereed)
    Abstract [en]

    The purpose of this study was to characterize associations between PINK1 genotypes, PINK1 transcript levels, and metabolic phenotypes in healthy adults and those with type 2 diabetes (T2D). We measured PINK1 skeletal muscle transcript levels and 8 independent PINK1 single nucleotide polymorphisms (SNPs) in a cohort of 208 Danish whites and in a cohort of 1701 British whites (SNPs and metabolic phenotypes only). Furthermore, we assessed the effects of PINK1 transcript ablation in primary adipocytes using RNA interference (RNAi). Six PINK1 SNPs were associated with PINK1 transcript levels (P < 0.04 to P < 0.0001). Obesity modified the association between PINK1 transcript levels and T2D risk (interaction P=0.005); transcript levels were inversely related with T2D in obese (n=105) [odds ratio (OR) per SD increase in expression levels=0.44; 95% confidence interval (CI): 0.23, 0.84; P=0.013] but not in nonobese (n=103) (OR=1.20; 95% CI: 0.82, 1.76; P=0.34) individuals. In the British cohort, several PINK1 SNPs were associated with plasma nonesterified fatty acid concentrations. Nominal genotype associations were also observed for fasting glucose, 2-h glucose, and maximal oxygen consumption, although these were not statistically significant after correcting for multiple testing. In primary adipocytes, Pink1 knockdown affected fatty acid binding protein 4 (Fabp4) expression, indicating that PINK1 may influence substrate metabolism. We demonstrate that PINK1 polymorphisms are associated with PINK1 transcript levels and measures of fatty acid metabolism in a concordant manner, whereas our RNAi data imply that PINK1 may indirectly influence lipid metabolism.

  • 5. Juks, Carmen
    et al.
    Lorents, Annely
    Arukuusk, Piret
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Pooga, Margus
    Cell-penetrating peptides recruit type A scavenger receptors to the plasma membrane for cellular delivery of nucleic acids2017In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 31, no 3, 975-988 p.Article in journal (Refereed)
    Abstract [en]

    Scavenger receptors (SRs) are a large family of multifunctional receptors that are involved in a range of physiologic and pathologic processes. The ability of class A scavenger receptors (SR-As) to bind anionic ligands facilitates the internalization of negatively charged cell-penetrating peptide (CPP)-nucleic acid nanocomplexes and thus makes them attractive targets for delivery of various nucleic acids. Recently, we demonstrated that SR-A3 and SR-A5 are recruited from intracellular membranes to the plasma membrane after incubation with PepFect 14-splice-switching oligonucleotide complexes. Here, we examined the mechanisms responsible for translocation of SR-As to the cell surface. We demonstrate that, in addition to nanocomplexes, some amphipathic CPPs are able to induce externalization of SR-A3 and SR-A5, and this process requires the presence of calcium ions. Furthermore, translocation of SR-A3 and SR-A5 requires activity of phosphatidylinositol-3-kinase, intact actin cytoskeleton, and the presence of serum proteins in culture medium.

  • 6.
    Kramarova, Tatiana V.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Shabalina, Irina G.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Andersson, Ulf
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Westerberg, Rolf
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Carlberg, Inger
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Houstek, Josef
    Nedergaard, Jan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Cannon, Barbara
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-Fo subunit P1 isoform2008In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, no 1, 55-63 p.Article in journal (Refereed)
    Abstract [en]

    Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP synthase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1-isoform of the c-F-o subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1-c subunit isoform under the promoter of the brown adipose tissue-specific protein UCP1. In the resulting UCP1p1 transgenic mice, total P1-c subunit mRNA levels were increased; mRNA levels of other F1F(o)-ATPase subunits were unchanged. In isolated brown-fat mitochondria, protein levels of the total c-Fo subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F-1-ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F1Fo-ATPase. Thus, the levels of the c-F-o subunit P1-isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c-F-o subunit may be a determining factor for F1Fo-ATPase assembly in all higher eukaryotes.-Kramarova, T. V., Shabalina, I. G., Andersson, U., Westerberg, R., Carlberg, I., Houstek, J., Nedergaard, J., Cannon, B. Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-F-o subunit P1 isoform.

  • 7. Lindholm, Malene E.
    et al.
    Huss, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Solnestam, Beata W.
    Kjellqvist, Sanela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundeberg, Joakim
    Sundberg, Carl J.
    The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing2014In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 10, 4571-4581 p.Article in journal (Refereed)
    Abstract [en]

    Human skeletal muscle health is important for quality of life and several chronic diseases, including type II diabetes, heart disease, and cancer. Skeletal muscle is a tissue widely used to study mechanisms behind different diseases and adaptive effects of controlled interventions. For such mechanistic studies, knowledge about the gene expression profiles in different states is essential. Since the baseline transcriptome has not been analyzed systematically, the purpose of this study was to provide a deep reference profile of female and male skeletal muscle. RNA sequencing data were analyzed from a large set of 45 resting human muscle biopsies. We provide extensive information on the skeletal muscle transcriptome, including 5 previously unannotated protein-coding transcripts. Global transcriptional tissue homogeneity was strikingly high, within both a specific muscle and the contralateral leg. We identified >23,000 known isoforms and found >5000 isoforms that differ between the sexes. The female and male transcriptome was enriched for genes associated with oxidative metabolism and protein catabolic processes, respectively. The data demonstrate remarkably high tissue homogeneity and provide a deep and extensive baseline reference for the human skeletal muscle transcriptome, with regard to alternative splicing, novel transcripts, and sex differences in functional ontology.

  • 8.
    Lundberg, Pontus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sütlü, Tolga
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Delivery of short interfering RNA using endosomolytic cell-penetrating peptides2007In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 21, no 11, 2664-2671 p.Article in journal (Refereed)
    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.

  • 9.
    Löfgren, Kajsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bedecs, Katarina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Antiprion properties of prion protein-derived cell-penetrating peptides2008In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, no 7, 2177-2184 p.Article in journal (Refereed)
    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.

  • 10.
    Morrison, Jamie I
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Borg, Paula
    Simon, András
    Plasticity and recovery of skeletal muscle satellite cells during limb regeneration2010In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 3, 750-756 p.Article in journal (Refereed)
    Abstract [en]

    Salamander limb regeneration depends on local progenitors whose progeny are recruited to the new limb. We previously identified a Pax7+ cell population in skeletal muscle whose progeny have the potential to contribute to the regenerating limb. However, the plasticity of individual Pax7+ cells, as well as their recovery within the new limb, was unclear. Here, we show that Pax7+ cells remain present after multiple rounds of limb amputation/regeneration. Pax7+ cells are found exclusively within skeletal muscle in the regenerating limb and proliferate where the myofibers are growing. Pax7 is rapidly down-regulated in the blastema, and analyses of clonal derivatives show that Pax7+ cell progeny are not restricted to skeletal muscle during limb regeneration. Our data suggest that the newt regeneration blastema is not entirely a composite of lineage-restricted progenitors. The results demonstrate that except for a transient and subsequently blunted increase, skeletal muscle satellite cells constitute a stable pool of reserve cells for multiple limb regeneration events.

  • 11.
    Palm-Apergi, Caroline
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lorents, Annely
    Padari, Kärt
    Pooga, Margus
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The membrane repair response masks membrane disturbances caused by cell-penetrating peptide uptake2009In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 23, no 1, 214-223 p.Article in journal (Refereed)
    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.

  • 12.
    Pooga, Margus
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Estonian Biocentre, Estonia.
    Kut, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Kihlmark, Madeleine
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Fernaeus, Sandra
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Raid, Raivo
    Land, Tiit
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Hallberg, Einar
    Bartfai, Tamas
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Scripps Research Institute, California.
    Cellular translocation of proteins by transportan2001In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 15, no 6, 1451-1453 p.Article in journal (Refereed)
    Abstract [en]

    Proteins with molecular masses ranging from 30 kDa. (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein-peptide constructs to penetrate into Bowes melanoma, BRL, and COS-7 cells. After 0.5-3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS-7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.

  • 13.
    Shabalina, Irina G.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Landreh, Luise
    Edgar, Daniel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Hou, Mi
    Gibanova, Natalia
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Atanassova, Nina
    Petrovic, Natasa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hultenby, Kjell
    Söder, Olle
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Svechnikov, Konstantin
    Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, no 8, 3274-3286 p.Article in journal (Refereed)
    Abstract [en]

    Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase gamma, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels. Although mtDNA mutator mice showed a dramatic reduction in testicle weight (only 15% remaining) and similar decreases in number of spermatozoa, testosterone levels in mt DNA mutator mice were unexpectedly fully unchanged. Leydig cell did not escape mitochondrial damage (only 20% of complex I and complex IV remaining) and did show high levels of reactive oxygen species (ROS) production (>5-fold increased), and permeabilized cells demonstrated absence of normal mitochondrial function. Nevertheless, within intact cells, mitochondrial membrane potential remained high, and testosterone production was maintained. This implies development of a compensatory mechanism. A rescuing mechanism involving electronsfrom the pentose phosphate pathway transferred via a 3-fold up-regulated cytochrome b5 to cytochrome c, allowing for mitochondrial energization, is suggested. Thus, the Leydig cells escape mitochondrial dysfunction via a unique rescue pathway. Such a pathway, bypassing respiratory chain dysfunction, may be of relevance with regard to mitochondrial disease therapy and to managing ageing in general.

  • 14. Tjellström, Henrik
    et al.
    Hellgren, Lars I.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sandelius, Anna Stina
    Lipid asymmetry in plant plasma membranes: phosphate deficiency-induced phospholipid replacement is restricted to the cytosolic leaflet2010In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 4, 1128-1138 p.Article in journal (Refereed)
    Abstract [en]

    As in other eukaryotes, plant plasma membranes contain sphingolipids, phospholipids, and free sterols. In addition, plant plasma membranes also contain sterol derivatives and usually <5 mol% of a galactolipid, digalactosyldiacylglycerol (DGDG). We earlier reported that compared to fully fertilized oats (Avena sativa), oats cultivated without phosphate replaced up to 70 mol% of the root plasma membrane phospholipids with DGDG. Here, we investigated the implications of a high DGDG content on membrane properties. The phospholipid-to-DGDG replacement almost exclusively occurred in the cytosolic leaflet, where DGDG constituted up to one-third of the lipids. In the apoplastic (exoplasmic) leaflet, as well as in rafts, phospholipids were not replaced by DGDG, but by acylated sterol glycosides. Liposome studies revealed that the chain ordering in free sterol/phospholipid mixtures clearly decreased when > 5mol% DGDG was included. As both the apoplastic plasma membrane leaflet (probably the major water permeability barrier) and rafts both contain only trace amounts of DGDG, we conclude that this lipid class is not compatible with membrane functions requiring a high degree of lipid order. By not replacing phospholipids site specifically with DGDG, negative functional effects of this lipid in the plasma membrane are avoided.

  • 15. Vukojević, Vladana
    et al.
    Ming, Yu
    D'Addario, Claudio
    Hansen, Mats
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Schulz, Rüdiger
    Johansson, Björn
    Rigler, Rudolf
    Terenius, Lars
    Mu-opioid receptor activation in live cells2008In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 22, no 10, 3537-3548 p.Article in journal (Refereed)
    Abstract [en]

    Interaction of the mu-opioid receptor (MOP) with selected ligands was investigated in live cells using advanced imaging by confocal laser scanning microscopy integrated with fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy. In PC12 cells stably transformed to express the fluorescently labeled MOP-enhanced green fluorescent protein construct, two pools of MOP were identified that could be discriminated by differences in their lateral mobility in the cell membrane. The majority of MOP receptors (80 +/- 10%) were characterized by a diffusion coefficient D-MOP,D-1 = (4 +/- 2) X 10(-11) m(2) s(-1), compared with the slowly moving fraction, D-MOP,D- 2 = (4 +/- 2) x 10(-12) m(2) s(-1). On stimulation with selected agonists ([D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin, enkephalin-heptapeptide Tyr-Gly-Gly-Phe-Met-Arg-Phe, morphine, and methadone), surface density of the MOP decreased, whereas the lateral mobility increased. In contrast, antagonists (naloxone and naltrexone) "froze" the receptor in the membrane, i.e., increased MOP surface density and decreased lateral mobility. Agonist activation was also accompanied by pronounced changes in the dynamics of plasma membrane lipids, as revealed by the general lipid marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate dye. The results provide new information about MOP activation in live cells at the molecular level, with a special focus on the dynamics of the intricate interplay between this receptor and the surrounding lipids.

  • 16.
    Waluk, Dominik P.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Schultz, Niklas
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Hunt, Mary C.
    Identification of glycine N-acyltransferase-like 2 (GLYATL2) as a transferase that produces N-acyl glycines in humans.2010In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 8, 2795-2803 p.Article in journal (Refereed)
    Abstract [en]

    The discovery of glycine conjugates of long-chain fatty acids (N-acyl glycines) in the brain and other non-neuronal tissues has led to the identification of an emerging class of bioactive lipids. The biological activities of N-acyl glycines include antinociceptive, anti-inflammatory and antiproliferative effects, and activation of G-protein-coupled receptors. However, despite the fact that N-acyl glycines are emerging as a distinct lipid signaling family, pathways for their production are not fully elucidated. Here we report on the characterization of human glycine N-acyltransferase-like 2 (hGLYATL2), a member of a gene family of 4 putative glycine conjugating enzymes, and show that it synthesizes various N-acyl glycines. Recombinantly expressed hGLYATL2 efficiently conjugated oleoyl-CoA, arachidonoyl-CoA, and other medium- and long-chain acyl-CoAs to glycine. The enzyme was specific for glycine as an acceptor molecule, and preferentially produced N-oleoyl glycine. The hGLYATL2 enzyme is localized to the endoplasmic reticulum, and the mRNA shows highest expression in salivary gland and trachea, but is also detected in spinal cord and skin fibroblasts. The expression pattern and the identification of high levels of N-acyl glycines in skin and lung may indicate a role for N-acyl glycines in barrier function/immune response and the potential role of hGLYATL2 in this regard is discussed.

  • 17.
    Zadravec, Damir
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Brolinson, Annelie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Fisher, Rachel
    Carneheim, Claes
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Csikasz, Robert
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Bertrand-Michel, Justine
    Borén, Jan
    Guillou, Hervé
    Rudling, Mats
    Jacobsson, Anders
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Ablation of the very long chain fatty acid elongase ELOVL3 in mice leads to constrained lipid storage and resistance to diet-induced obesity2010In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 24, no 11, 4366-4377 p.Article in journal (Refereed)
    Abstract [en]

    Although saturated and monounsaturated very-long-chain fatty acids (VLCFA) have long been associated with undesirable effects on health, including obesity, heart failure and atherosclerosis, the physiological role of endogenous synthesis is largely unknown. The fatty acid elongase ELOVL3 is involved in the synthesis of C20-C24 saturated and monounsaturated VLCFA mainly in liver, brown and white adipose tissue and in triglyceride rich glands such as the sebaceous and meibomian glands. Here we show that ablation of ELOVL3 leads to reduced adiponectin levels, constrained expansion of adipose tissue and resistance against diet-induced obesity, a situation that is more exaggerated in female mice. Both female and male knockout mice show reduced hepatic lipogenic gene expression and triglyceride content, a situation, which is associated with, reduced expression of PPARg and its target genes. As a consequence, the VLDL-triglyceride level in serum is significantly reduced. Remarkably, despite increased energy expenditure, markedly reduced serum levels of leptin and increased expression of orexigenic peptides in the hypothalamus, the Elovl3-/- mice do not compensate by increased food intake. Thus, these results reveal that C20-C22 saturated and monounsaturated VLCFA produced by ELOVL3 are indispensable for appropriate synthesis of liver triglycerides, fatty acid uptake and storage in adipose tissue.

  • 18. Zingaretti, Maria Cristina
    et al.
    Crosta, Francesca
    Vitali, Alessandra
    Guerrieri, Mario
    Frontini, Andrea
    Cannon, Barbara
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Physiology.
    Cinti, Saverio
    The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue.2009In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 23, no 9, 3113-20 p.Article in journal (Refereed)
    Abstract [en]

    Classically, adult humans have been considered not to possess active brown adipose tissue (BAT). However, positron-emission-tomography has shown fluorodeoxyglucose uptake that is distributed in such a way (e.g., in the neck) that it would seem to be BAT. Until now this has not been supported by direct evidence that these areas truly represented BAT, that is, the presence of the BAT-unique uncoupling protein-1 (UCP1). Samples of adipose tissue from the neck of 35 patients undergoing surgery for thyroid diseases were obtained and analyzed. In 1/3 of the subjects (the younger and leaner), distinct islands composed of UCP1 immunoreactive brown adipocytes could clearly be discerned, accounting for up to 1/3 of all adipocytes. The brown-adipose islands were richly sympathetically innervated (indicating acute central control); adjacent white adipose areas were not. The capillary density was high, implying a high capacity for oxygen delivery. Cells with features of brown adipocyte precursors were found in pericapillary areas. These data demonstrate that human adults indeed possess BAT and thus imply possibilities of future therapeutic strategies for the treatment of obesity, including maintenance of brown adipocytes and stimulation of the growth of preexisting brown precursors.

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