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  • 1.
    Abdo, R.A.
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sellerholm, Alexander
    Stockholm University, Faculty of Science, Department of Physics.
    Conrad, Jan
    Stockholm University, Faculty of Science, Department of Physics.
    Spectrum of the Isotropic Diffuse Gamma-Ray Emission Derived from First-Year Fermi Large Area Telescope Data2010In: Physical Review Letters, ISSN 0031-9007, Vol. 104, no 101101Article in journal (Refereed)
    Abstract [en]

    We report on the first Fermi Large Area Telescope (LAT) measurements of the so-called ‘‘extra- galactic’’ diffuse gamma-ray emission (EGB). This component of the diffuse Υ-ray emission is generally considered to have an isotropic or nearly isotropic distribution on the sky with diverse contributions discussed in the literature. The derivation of the EGB is based on detailed modeling of the bright foreground diffuse Galactic gamma-ray emission, the detected LAT sources, and the solar gamma-ray emission. We find the spectrum of the EGB is consistent with a power law with a differential spectral index of 2.41 \pm 0.05 and intensity I(>100 MeV)=(1.03\pm 0.17) e-5 cm**2 s **-1 sr**-1 , where the error is systematics dominated. Our EGB spectrum is featureless, less intense, and softer than that derived from EGRET data.

  • 2.
    Adlerz, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Processing of the amyloid precursor protein and its paralogues amyloid precursor-like proteins 1 and 22007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Alzheimer’s disease (AD) is a neurodegenerative disorder which is histopathologically characterised by amyloid plaques and neurofibrillary tangles. Amyloid plaques consist of the amyloid β-peptide (Aβ) that can form aggregates in the brain. Aβ is generated from the amyloid precursor protein (APP) through proteolytic cleavage. APP belongs to a conserved protein family that also includes the two paralogues, APP-like proteins 1 and 2 (APLP1 and APLP2). Despite the immense amount of research on APP, motivated by its implication in AD, the function of this protein family has not yet been determined. In this thesis, we have studied the expression and proteolytic processing of the APP protein family. Our results are consistent with previous findings that suggest a role for APP during neuronal development. Treatment of cells with retinoic acid (RA) resulted in increased synthesis. In addition, we observed that RA treatment shifted the processing of APP from the amyloidogenic to the non-amyloidogenic pathway. The proteins in the APP family have been hard to distinguish both with respect to function and proteolytic processing. However, for development of new drugs with APP processing enzymes as targets this is of great importance. Our studies suggest similarities, but also differences in the mechanism regulating the processing of the different paralogues. We found that brain-derived neurotrophic factor (BDNF) had different impact on the members of the APP family. Most interestingly, we also found that the mechanism behind the increased processing in response to IGF-1 was not identical between the homologous proteins. In summary, our results indicate that in terms of regulation APLP1 and APLP2 differ more from each other than from APP. Our studies open up the possibility of finding means to selectively block Aβ production without interfering with the processing and function of the paralogous proteins.

  • 3.
    Adlerz, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holback, Sofia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Multhaup, Gerd
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    IGF-1-induced Processing of the Amyloid Precursor Protein Family Is Mediated by Different Signaling Pathways2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 14, 10203-10209 p.Article in journal (Refereed)
    Abstract [en]

    The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.

  • 4.
    Ajayi, Abiodun
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Molecular mechanism(s) underlying neurodegeneration in SCA7 disease: Role of NOX enzymes and oxidative stress2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide expansion in the SCA7 gene resulting in progressive ataxia and retinal dystrophy. SCA7 belongs to a group of neurodegenerative disorders called polyglutamine (polyQ) diseases, that share the common feature of glutamine tract expansions within otherwise unrelated proteins. Common suggested mechanisms by which polyQ expanded proteins induce toxicity include aggregation and induction of oxidative stress. 

    In this work we examined the connection between oxidative stress, aggregation and toxicity in SCA7 disease. We show that expression of the SCA7 disease protein, ataxin-7 (ATXN7), results in elevated levels of ROS and oxidative stress which in turn lead to toxicity. Our results also revealed that the oxidative stress further contributes to mutant ATXN7 aggregation. Moreover, we show, for the first time, that the major source of the elevated ROS in mutant ATXN7 cells is the increased activation of NOX1 enzymes. Interestingly, our results further revealed that the increased level of NOX1 activity together with altered p53 function leads to a metabolic shift in mutant ATXN7 expressing cells. Treatments with antioxidants, a NOX1 specific inhibitor or NOX1 knock-down, all decreased the ROS level, restored the metabolic shift and ameliorated the mutant ATXN7 induced toxicity. Taken together, we conclude that mutant ATXN7 activate NOX1 enzymes which results in oxidative stress, increased mutant ATXN7 aggregation, metabolic dysfunction and toxicity. NOX1 specific inhibition could thus be a potential therapeutic strategy for SCA7.

  • 5.
    Ajayi, Abiodun
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Study of molecular mechanism(s) underlying neurodegeneration in SCA7 disease: Role of NOX enzymes and oxidative stress2013Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide expansion in the SCA7/ATXN7 gene resulting in progressive ataxia and retinal dystrophy. SCA7 belongs to a group of neurodegenerative disorders called polyglutamine (polyQ) diseases, that share the common feature of glutamine tract expansions within otherwise unrelated proteins. Common suggested mechanisms by which polyQ disorders induce toxicity include aggregation and induction of oxidative stress.

    In this work, we examined the connection between oxidative stress and toxicity in SCA7 disease. We showed that expression of mutant ataxin-7 (ATXN7) results in elevated level of reactive oxygen species (ROS) and oxidative stress, leading to toxicity. Our results also revealed that the oxidative stress further contributes to mutant ATXN7 aggregation. We showed, for the first time, that the source of the ROS in mutant ATXN7 cells is thorough the activation of the NOX1 enzyme. Interestingly, our results further revealed that the increased level of NOX1 activity and expression by mutant ATXN7 results in a metabolic shift similar to the Warburg effect. Treatments with antioxidants or a NOX1 specific inhibitor decreased the ROS level, restored the metabolic shift and ameliorated the ATXN7 induced toxicity. Taken together, we suggest that mutant ATXN7 specifically activate NOX1 enzyme and that antioxidants treatment or NOX1 specific inhibition could be a potential therapeutic strategy for SCA7.

  • 6.
    Ajayi, Abiodun
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Karlström, Victor
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    NOX1 and p53 cross-talk in SCA7 polyglutamine toxicityManuscript (preprint) (Other academic)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is one of nine neurodegenerative disorders caused by expanded polyglutamine repeats. Common toxic gain-of-function mechanisms, including oxidative stress and metabolic dysfunction, have been proposed in these disorders. In a recent study we identified increased activity of the ROS producing NADPH oxidase 1 (NOX1) enzyme and reduced activity of the p53 transcription factor as contributing factors to the oxidative stress and metabolic dysfunction in a SCA7 model. In this study we further investigate the molecular mechanisms behind the altered NOX1 and p53 activity, as well as how these two molecules cross-talk to promote oxidative stress, metabolic dysfunction and toxicity in SCA7. We show that increased NOX1 protein stability, as well as alteration of p53-mediated regulation of NOX1 mRNA levels, contributes to the elevated NOX1 expression in SCA7 cells. Furthermore, we show that the enhance NOX1 activity in SCA7 cells is associated with increased oxidation of p53 and promotes a shift in the p53 sub-cellular localization, as well reduction of soluble p53 levels. Taken together, our results suggest that in SCA7 cells a feed-forward loop between NOX1 and p53 is induced. In this loop NOX1-mediated p53 oxidation results in altered p53 localization and reduced p53 transcriptional activity. In turn, the reduced p53 transcriptional activity promotes the activation of NOX1 mRNA and activity. This loop then contributes to the metabolic dysregulation, oxidative stress and toxicity in SCA7 cells.

  • 7.
    Ajayi, Abiodun
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Yu, Xin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Expanded ataxin-7 cause toxicity by inducing ROS production from NADPH oxidase complexes in a stable inducible Spinocerebellar ataxia type 7 (SCA7) model2012In: BMC neuroscience (Online), ISSN 1471-2202, Vol. 13, 86Article in journal (Refereed)
    Abstract [en]

    Background: Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common mechanisms of disease pathogenesis suggested for polyQ disorders include aggregation of the polyQ protein and induction of oxidative stress. However, the exact mechanism(s) of toxicity is still unclear. Results: In this study we show that expression of polyQ expanded ATXN7 in a novel stable inducible cell model first results in a concomitant increase in ROS levels and aggregation of the disease protein and later cellular toxicity. The increase in ROS could be completely prevented by inhibition of NADPH oxidase (NOX) complexes suggesting that ATXN7 directly or indirectly causes oxidative stress by increasing superoxide anion production from these complexes. Moreover, we could observe that induction of mutant ATXN7 leads to a decrease in the levels of catalase, a key enzyme in detoxifying hydrogen peroxide produced from dismutation of superoxide anions. This could also contribute to the generation of oxidative stress. Most importantly, we found that treatment with a general anti-oxidant or inhibitors of NOX complexes reduced both the aggregation and toxicity of mutant ATXN7. In contrast, ATXN7 aggregation was aggravated by treatments promoting oxidative stress. Conclusion: Our results demonstrates that oxidative stress contributes to ATXN7 aggregation as well as toxicity and show that anti-oxidants or NOX inhibition can ameliorate mutant ATXN7 toxicity.

  • 8.
    Ajayi, Abiodun
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Yu, Xin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The role of NADPH oxidase (NOX) enzymes in neurodegenerative disease2013In: Frontiers in Biology, ISSN 1674-7992, Vol. 8, no 2, 175-188 p.Article in journal (Refereed)
    Abstract [en]

    Recently, mounting evidence implicating reactive oxygen species (ROS) generated by NADPH oxidase(NOX) enzymes in the pathogenesis of several neurodegenerative diseases including Amyotrophic lateral sclerosis(ALS), Alzheimer’s (AD), Parkinson’s (PD) and polyglutamine disease, have arisen. NOX enzymes are transmembraneproteins and generate reactive oxygen species by transporting electrons across lipid membranes. Under normal healthyconditions, low levels of ROS produced by NOX enzymes have been shown to play a role in neuronal differentiation andsynaptic plasticity. However, in chronic neurodegenerative diseases over-activation of NOX in neurons, as well as inastrocytes and microglia, has been linked to pathogenic processes such as oxidative stress, exitotoxicity andneuroinflammation. In this review, we summarize the current knowledge about NOX functions in the healthy centralnervous system and especially the role of NOX enzymes in neurodegenerative disease processes.

  • 9.
    Ajayi, Abiodun
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Yu, Xin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Wahlo-Svedin, Carolina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Polyglutamine expanded ataxin-7 alters NOX1 activity and cellular metabolism2013Article in journal (Refereed)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is one of nine inherited neurodegenerative disorders caused by polyglutamine (polyQ) expansions. Common pathogenic mechanisms, including oxidative stress and metabolic dysfunction, have been implicated in polyQ disease. However, the exact toxic mechanism(s) is still unclear. We have previously demonstrated that expression of the SCA7 disease protein, ATXN7, results in oxidative stress and toxicity via activation of ROS-producing NADPH oxidase (NOX) enzymes. In this study, we show that mutant ATXN7 specifically up-regulates and activates the NOX1 family member. Furthermore, we show that the increased NOX1 activity is linked with a metabolic shift, similar to the Warburg effect, and reduced energy levels. Reduction of the NOX1-mediated ROS production reverse the metabolic shift and rescue the ATXN7 induced toxicity. These data suggest that NOX1-mediated metabolic alterations and energy deficit could play a role in SCA7 pathology and possibly in other polyQ diseases.

  • 10.
    Ajayi, Abiodun
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Yu, Xin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Wahlo-Svedin, Carolina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Tsirigotaki, Galateia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Karlström, Victor
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ström, Anna-Lena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Altered p53 and NOX1 activity cause bioenergetic defects in a SCA7 polyglutamine disease model2015In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1847, no 4-5, 418-428 p.Article in journal (Refereed)
    Abstract [en]

    Spinocerebellar ataxia type 7 (SCA7) is one of the nine neurodegenerative disorders caused by expanded polyglutamine (polyQ) domains. Common pathogenic mechanisms, including bioenergetics defects, have been suggested for these so called polyQ diseases. However, the exact molecular mechanism(s) behind the metabolic dysfunction is still unclear. In this study we identified a previously unreported mechanism, involving disruption of p53 and NADPH oxidase 1 (NOX1) activity, by which the expanded SCA7 disease protein ATXN7 causes metabolic dysregulation. The NOX1 protein is known to promote glycolytic activity, whereas the transcription factor p53 inhibits this process and instead promotes mitochondrial respiration. In a stable inducible PC12 model of SCA7, p53 and mutant ATXN7 co-aggregated and the transcriptional activity of p53 was reduced, resulting in a 50% decrease of key p53 target proteins, like AIF and TIGAR. In contrast, the expression of NOX1 was increased approximately 2 times in SCA7 cells. Together these alterations resulted in a decreased respiratory capacity, an increased reliance on glycolysis for energy production and a subsequent 20% reduction of ATP in SCA7 cells. Restoring p53 function, or suppressing NOX1 activity, both reversed the metabolic dysfunction and ameliorated mutant ATXN7 toxicity. These results hence not only enhance the understanding of the mechanisms causing metabolic dysfunction in SCA7 disease, but also identify NOX1 as a novel potential therapeutic target in SCA7 and possibly other polyQ diseases.

  • 11. Alier, Kwai
    et al.
    Chen, Yishen
    Eriksson Sollenberg, Ulla
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Smith, Peter
    Selective stimulation of GalR1 and GalR2 in rat substantia gelatinosa reveals a cellular basis for the anti- and pro-nociceptive actions of galanin2008In: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 137, no 1, 138-146 p.Article in journal (Refereed)
    Abstract [en]

    Galanin modulates spinal nociceptive processing by interacting with two receptors, GalR1 and GalR2. The underlying neurophysiological mechanisms were examined by whole-cell recording from identified neurons in the substantia gelatinosa of young adult rats. GalR1 was activated with a 'cocktail' containing the GalR1/2 agonist, AR-M 961 (0.5 mu M), in the presence of the GalR2 antagonist, M871 (1.0-2.5 mu M). GalR2 was activated with the selective agonist, AR-M 1896 (0.5-1.0 mu M). Application of the 'GalR1 agonist cocktail' often activated an inwardly-rectifying conductance in delay firing (excitatory) and tonically firing (inhibitory) neurons. This conductance was not activated by AR-M 1896 which instead decreased or increased an outwardly-rectifying conductance at voltages positive to -70 rnV. Despite this variability in its actions on current-voltage relationships, AR-M 1896 very consistently decreased membrane excitability, as measured by cumulative action potential latency in response to a depolarizing current ramp. This strong GalR2-mediated effect was seen in neurons where membrane conductance was decreased, and where membrane excitability might be predicted to increase. GalR2 was also located presynaptically, as AR-M 1896 increased the interevent interval of spontaneous EPSCs in both delay and tonic cells. By contrast, the 'GalR1 agonist cocktail' had little effect on spontaneous EPSCs, suggesting that presynaptic terminals do not express GalR1. These diverse actions of GalR1 and GalR2 activation on both inhibitory and excitatory neurons are discussed in relation to the known spinal antinociceptive and pro-nociceptive actions of galanin, to the possible association of GalR1 with the inhibitory G-protein, G(i/o) and to report that GalR2 activation suppresses Ca(2+) channel currents.

  • 12.
    Amelina, Hanna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holm, Tina
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Delivering catalase to yeast peroxisomes using cell-penetrating peptidesIn: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658Article in journal (Refereed)
  • 13. Anderson, Maria E.
    et al.
    Runesson, Johan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Saar, Indrek
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Robinson, John K.
    Galanin, through GalR1 but not GalR2 receptors, decreases motivation at times of high appetitive behavior2013In: Behavioural Brain Research, ISSN 0166-4328, E-ISSN 1872-7549, Vol. 239, 90-93 p.Article in journal (Refereed)
    Abstract [en]

    Galanin is a 29/30-amino acid long neuropeptide that has been implicated in many physiological and behavioral functions. Previous research has shown that i.c.v. administration of galanin strongly stimulates food intake in sated rats when food is freely available, but fails to stimulate this consumption when an operant response requirement is present. Using fixed ratio (FR) schedules, we sought to further clarify galanin's role in motivated behavior by administering galanin i.c.v. to rats working on fixed ratio schedules requiring either a low work condition (FR1) or higher work conditions (FR > 1) to obtain a 0.2% saccharin reward. Rats in the FR > 1 group were assigned to either an FR3, FR5 or FR7 schedule of reinforcement. The rate of reinforcement decreased for only the FR > 1 group as compared to saline controls. Furthermore, injections of GalR1 receptor agonist M617 led to a similar, marginally significant decrease in the number of reinforcers received in the FR > 1 condition, but a decrease was not seen after injections of GalR2 receptor agonist M1153. Taken together, these results show that galanin may be playing a role in decreasing motivation at times of high appetitive behavior, and that this effect is likely mediated by the GalR1 receptor.

  • 14.
    Andres, M I
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Forsby, A
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Walum, E
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Polygodial-induced noradrenaline release in human neuroblastoma SH-SY5Y cells.1997In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 11, no 5, 509-11 p.Article in journal (Refereed)
    Abstract [en]

    Polygodial is a natural sesquiterpene which exhibits pronounced pungency and a powerful antifeedant activity. At low concentrations, which do not alter general cell membrane permeability, polygodial increases the intracellular concentration of free calcium ([Ca(2+)](i)). Sensory neurotransmission depends on noradrenaline (NA) release, and vesicular exocytosis, in turn, is dependent on an increase in [Ca(2+)](i). The nociceptive response induced by polygodial could therefore be directly linked to intracellular calcium levels. Consequently, the objective of this work was to investigate the effect of polygodial on NA release. The human neuroblastoma cell line SH-SY5Y was selected as an in vitro model for sensory neurones. Semiconfluent cells were preloaded with tritiated NA ([(3)H]NA). After 3 min exposure of polygodial to the cells, released and unreleased radioactivity were measured. Polygodial induced a significant [(3)H]NA release at concentrations between 0.1 and 0.5 mug/ml with a maximum effect at 0.2 mug/ml (40% increased release of [(3)H]NA as compared with unstimulated control cells). No polygodial-induced transmitter release was seen at 3.5 and 5 mug/ml. For comparison, carbachol (1 rim) increased [(3)H]NA release by 10% and the KCl-induced (100 mm) [(3)H]NA release increased by 8% as compared with unstimulated SH-SY5Y cells. In conclusion polygodial, at the concentrations 0.1-0.5 mug/ml (equal to 0.4-2 mum), induces NA release which is dependent on polygodial-induced increase in [Ca(2+)](i).

  • 15. Anko, Maja
    et al.
    Majhenc, Janja
    Kogej, Ksenija
    Sillard, Rannard
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Anderluh, Gregor
    Zorko, Matjaz
    Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane2012In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1818, no 3, 915-924 p.Article in journal (Refereed)
    Abstract [en]

    The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PR and PF6 observed in previous studies.

  • 16. Arabi, Azadeh
    et al.
    Rustum, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Wright, Anthony P. H.
    Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels2003In: Journal of Cell Science, ISSN 0021-9533, Vol. 116, no 9, 1707-1717 p.Article in journal (Refereed)
  • 17.
    Arrighi, Romanico B. G.
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Ebikeme, Charles
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ranford-Cartwright, Lisa
    Barrett, Michael P.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Cell-penetrating peptide TP10 shows broad-spectrum activity against both Plasmodium falciparum and Trypanosoma brucei brucei2008In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 52, no 9, 3414-3417 p.Article in journal (Refereed)
    Abstract [en]

    Malaria and trypanosomiasis are diseases which afflict millions and for which novel therapies are urgently required. We have tested two well-characterized cell-penetrating peptides (CPPs) for antiparasitic activity. One CPP, designated TP10, has broad-spectrum antiparasitic activity against Plasmodium falciparum, both blood and mosquito stages, and against blood-stage Trypanosoma brucei brucei.

  • 18. Arsov, Zoran
    et al.
    Nemec, Marjana
    Schara, Milan
    Johansson, Henrik
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zorko, Matjaž
    Cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes2008In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 14, no 12, 1303-1308 p.Article in journal (Refereed)
    Abstract [en]

    Interaction of the cell-penetrating peptide (CPP) cysteine-transportan (Cys-TP) with model lipid membranes was examined by spin-label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys-TP on membrane structure Was studied. The influence of Cys-TP on membrane permeability was monitored by the reduction of a liposome-trapped water-soluble spin probe. Cys-TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys-TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so-called liquid-ordered phase, no effect. of Cys-TP was observed, either on Line membrane structure or on the membrane permeability. The interaction between Cys-TP and the lipid membrane therefore depends on the lipid phase. This could be of great. importance for understanding of the CPP-lipid interaction in laterally heterogeneous membranes, white it implies that the CPP-lipid interaction can be different at different points along the membrane.

  • 19.
    Arukuusk, Piret
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Paernaste, Ly
    Margus, Helerin
    Eriksson, N. K. Jonas
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Vasconcelos, Luis
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Padari, Kaert
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Differential Endosomal Pathways for Radically Modified Peptide Vectors2013In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 10, 1721-1732 p.Article in journal (Refereed)
    Abstract [en]

    In the current work we characterize the uptake mechanism of two NickFect family members, NF51 and NF1, related to the biological activity of transfected plasmid DNA (pDNA). Both vectors condense pDNA into small negatively charged nanoparticles that transfect He La cells with equally high efficacy and the delivery is mediated by SCARA3 and SCARA.5 receptors. NF1 condenses DNA into less homogeneous and less stable nanoparticles than NF51. NF51/pDNA nanoparticles enter the cells via macropinocytosis, while NF1/pDNA complexes use clathrin- or caveolae-mediated endocytosis and macropinocytosis. Analysis of separated endosomal compartments uncovered lysomotropic properties of NF51 that was also proven by cotransfection with chloroquine. In summary we characterize how radical modifications in peptides, such as introducing a kink in the structure of NF51 or including extra negative charge by phospho-tyrosine substitution in NF1, resulted in equally high efficacy for gene delivery, although this efficacy is achieved by using differential transfection pathways.

  • 20. Arukuusk, Piret
    et al.
    Paernaste, Ly
    Oskolkov, Nikita
    Copolovici, Dana-Maria
    Margus, Helerin
    Padari, Kaert
    Moell, Kaidi
    Maslovskaja, Julia
    Tegova, Radi
    Kivi, Gaily
    Tover, Andres
    Pooga, Margus
    Ustav, Mart
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids2013In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 5, 1365-1373 p.Article in journal (Refereed)
    Abstract [en]

    Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

  • 21. Arukuusk, Piret
    et al.
    Pärnaste, Ly
    Hällbrink, Mattias
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. Tartu University, Estonia.
    PepFects and NickFects for the Intracellular Delivery of Nucleic Acids2015In: Cell-Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Springer, 2015, Vol. 1324, 303-315 p.Chapter in book (Refereed)
    Abstract [en]

    Nucleic acids can be utilized in gene therapy to restore, alter, or silence gene functions. In order to reveal the biological activity nucleic acids have to reach their intracellular targets by passing through the plasma membrane, which is impermeable for these large and negatively charged molecules. Cell-penetrating peptides (CPPs) condense nucleic acids into nanoparticles using non-covalent complexation strategy and mediate their delivery into the cell, whereas the physicochemical parameters of the nanoparticles determine the interactions with the membranes, uptake mechanism, and subsequent intracellular fate. The nanoparticles are mostly internalized by endocytosis that leads to the entrapment of them in endosomal vesicles. Therefore design of new CPPs that are applicable for non-covalent complex formation strategy and harness endosomolytic properties is highly vital. Here we demonstrate that PepFects and NickFects are efficient vectors for the intracellular delivery of various nucleic acids.This chapter describes how to form CPP/pDNA nanoparticles, evaluate stable nanoparticles formation, and assess gene delivery efficacy.

  • 22. Aschner, Michael
    et al.
    Levin, Edward D.
    Suñol, Cristina
    Olopade, James O.
    Helmcke, Kirsten J.
    Avila, Daiana S.
    Sledge, Damiyon
    Ali, Rahim H.
    Upchurch, Lucia
    Donerly, Susan
    Linney, Elwood
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Ponnuru, Padmavathi
    Connor, James R.
    Gene-environment interactions: Neurodegeneration in non-mammals and mammals2010In: Neurotoxicology, ISSN 0161-813X, E-ISSN 1872-9711, Vol. 31, no 5, 582-8 p.Article in journal (Refereed)
    Abstract [en]

    The understanding of how environmental exposures interact with genetics in central nervous system dysfunction has gained great momentum in the last decade. Seminal findings have been uncovered in both mammalian and non-mammalian model in large result of the extraordinary conservation of both genetic elements and differentiation processes between mammals and non-mammalians. Emerging model organisms, such as the nematode and zebrafish have made it possible to assess the effects of small molecules rapidly, inexpensively, and on a miniaturized scale. By combining the scale and throughput of in vitro screens with the physiological complexity and traditional animal studies, these models are providing relevant information on molecular events in the etiology of neurodegenerative disorders. The utility of these models is largely driven by the functional conservation seen between them and higher organisms, including humans so that knowledge obtained using non-mammalian model systems can often provide a better understanding of equivalent processes, pathways, and mechanisms in man. Understanding the molecular events that trigger neurodegeneration has also greatly relied upon the use of tissue culture models. The purpose of this summary is to provide-state-of-the-art review of recent developments of non-mammalian experimental models and their utility in addressing issues pertinent to neurotoxicity (Caenorhabditis elegans and Danio rerio). The synopses by Aschner and Levin summarize how genetic mutants of these species can be used to complement the understanding of molecular and cellular mechanisms associated with neurobehavioral toxicity and neurodegeneration. Next, studies by Suñol and Olopade detail the predictive value of cultures in assessing neurotoxicity. Suñol and colleagues summarize present novel information strategies based on in vitro toxicity assays that are predictive of cellular effects that can be extrapolated to effects on individuals. Olopade and colleagues describe cellular changes caused by sodium metavanadate (SMV) and demonstrate how rat primary astrocyte cultures can be used as predicitive tools to assess the neuroprotective effects of antidotes on vanadium-induced astrogliosis and demyelination.

  • 23.
    Aslam, Muhammad
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The fruit fly Drosophila melanogaster GSTE6 and E7; characterization, immobilization and transgenic overexpression2014Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Glutathione transferases (GSTs) are multifunctional enzymes that are universally distributed in most eukaryotes and prokaryotes. They play a pivotal role in the metabolism and detoxication of numerous endogenous and exogenous electrophiles by conjugating them with ubiquitous tripeptide glutathione. In this study we have immobilized two heterologously expressed and purified Epsilon-class enzymes, GSTE6 and GSTE7, from of Drosophila melanogaster on nanoporous alumina membranes. The membranes were derivatized with 3-aminopropyl-triethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzymes in solution. The stability of the immobilized enzymes was virtually identical to that for the enzymes in solution and no leakage of enzyme from the matrix could be observed.

    Additionally, we have investigated the catalytic activities of GSTE7 with organic isothiocyanates (ITCs). These reactive compounds are strong electrophilic molecules produced in plants by the hydrolysis of glucosinolates and exert toxicity in biological tissues.  Our in vitro studies, showed high catalytic activity of GSTE7 towards ITCs. We have then explored the in vivo effect of phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC) in transgenic fruit flies overexpressing GSTE7. A concentration of 0.25 mM PEITC in standard fly food was shown to be toxic to flies and significantly shortened the lifespan. We noticed that overexpression of GSTE7 could protect females from the initial acute toxic effects, but had no positive effect on long term exposure. The effect on males seems to be the opposite to that of females, where a higher mortality was seen in fly males overexpressing GST E7 after one week of exposure.  On the other hand 1mM concentration of AITC showed no toxic effects, but dramatically reduced the oviposition activity of wild-type flies in comparison to the transgenic flies.

  • 24.
    Aslam, Muhammad
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Dahlberg, Olle
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Mannervik, Mattias
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Transgenic Overexpression of Glutathione Transferase E7 in Drosophila Attenuates Toxicity of Organic Isothiocyanates Affecting Survival and OvipositionManuscript (preprint) (Other academic)
    Abstract [en]

    Organic isothiocyanates (ITCs) are allelochemicals produced by plants in order to combat insects and other herbivores. The compounds are toxic electrophiles that can be inactivated and conjugated with intracellular glutathione in reactions catalyzed by glutathione transferases (GSTs). The Drosophila melanogaster GSTE7 was heterologously expressed in Escherichia coli and purified for functional studies. The enzyme showed high catalytic activity with various isothiocyanates including phenethyl isothiocyanate (PEITC) and allyl isothiocyanate (AITC), which in millimolar dietary concentrations conferred toxicity to adult D. melanogaster leading to death or a shortened life-span of the flies. In situ hybridization revealed a maternal contribution of GSTE7 transcripts to embryos, and strongest zygotic expression in the digestive tract.  Transgenesis involving the GSTE7 gene controlled by an actin promoter produced viable flies expressing the GSTE7 transcript ubiquitously. Transgenic females show a significant extension in life-span when subjected to the same PEITC treatment as the wild-type flies. By contrast, transgenic male flies showed no significant effect in the first few days, and subsequently showed a somewhat lower survival rate. At 1 mM AITC concentration, no toxicity was noted. However, the oviposition activity was dramatically enhanced from a very low level in wild-type flies reared in the presence of 1 mM AITC to values an order of magnitude higher for the transgenic flies. The results demonstrate a clear protective effect of GSTE7 against exposure to ITC allelochemicals which can affect both life-span and fecundity of female flies.

  • 25.
    Attoff, Kristina
    Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm University.
    In vitro developmental neurotoxicity of acrylamide2016Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The number of children with neurodevelopmental disorders is increasing worldwide which makes it a public concern. Exposure to environmental chemicals has been reported as a source of developmental neurotoxicity. There is also an increase in the number of chemicals reaching the global market each year and currently there are thousands of substances that have not yet been tested for developmental neurotoxicity. The current developmental neurotoxicity testing guidelines are time consuming, expensive, require a lot of animals and have relatively low sensitivity understanding for the mechanisms of toxicology. The field of developmental neurotoxicity testing is in need of a paradigm shift to the use of alternative in vitro methods capable of testing and screening large number of substances. The next generation developmental neurotoxicity testing will consist of both in silico and in vitro testing that has to be used in a combined fashion so that it will generate a more rapid and efficient toxicity testing. The methods need to be standardized between laboratories so that reproducible data can be obtained. Simple endpoints will simply not be enough for in vitro developmental neurotoxicity testing models. Rather, a battery of more refined endpoints that pinpoints the specific toxicity of a compound, discriminate between different neural subpopulations and different stages of neural differentiation is crucial for success. The use of mRNA biomarkers could be a good example of such an endpoint, and have been suggested to be valuable in detecting developmental neurotoxicity. This thesis will give a broad overview of different alternative in vitro models for developmental neurotoxicity. Developmental neurotoxicity of acrylamide was investigated by using selected cell models and endpoints. Acrylamide is a well-known neurotoxic compound and most people get exposed to the compound by food consumption and from environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed and the risk for adverse effects in the developing nervous system is overwhelming. The neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as indicators for developmental neurotoxicity. The reduced neurite outgrowth in the SH-SY5Y cell model occurred at up to seven orders of magnitude lower than what have been previously shown for different neural cell systems. Acrylamide also affected the differentiation process in both neurons and glia cells in the C17.2 cell line. We show that acrylamide attenuated neural differentiation at seven orders of magnitude lower concentrations than the estimated plasma concentration of free acrylamide in the fetus. The fact that low concentrations seem to delay the differentiation process in both cell lines, raises cause for an alarm for developmental neurotoxicity induced by acrylamide.  

  • 26.
    Attoff, Kristina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kertika, Dimitra
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundqvist, Jessica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oredsson, S.
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm Univ, Dept Neurochem, S-10691 Stockholm, Sweden.
    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y2016In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 35, 100-111 p.Article in journal (Refereed)
    Abstract [en]

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10 fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10 pM. Acrylamide significantly reduced the number of neurons starting at 1 mu M and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

  • 27.
    Attoff, Kristina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm University.
    Kertika, Dimitra
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundqvist, Jessica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oredsson, Stina
    Lund University.
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5YManuscript (preprint) (Other academic)
  • 28.
    Axelsson, V
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pikkarainen, K
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Forsby, A
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells.2006In: Cell Biology and Toxicology, ISSN 0742-2091, E-ISSN 1573-6822, Vol. 22, no 2, 127-36 p.Article in journal (Refereed)
    Abstract [en]

    Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 micromol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with L: -buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of gamma-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.

  • 29.
    Axelsson, Viktoria
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Evaluation of neurotoxic properties of gliotoxin2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows:

    i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status.

    ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo.

    iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated.

    iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity.

    v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites.

    vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

  • 30.
    Axelsson, Viktoria
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Holback, Sofia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sjögren, Maria
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gustafsson, Helena
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gliotoxin induces caspase-dependent neurite degeneration and calpain-mediated general cytotoxicity in differentiated human neuroblastoma SH-SY5Y cells.2006In: Biochem Biophys Res Commun, ISSN 0006-291X, Vol. 345, no 3, 1068-74 p.Article in journal (Refereed)
    Abstract [en]

    In this study, a significant increase by 50% in intracellular free calcium concentration ([Ca(2+)](i)) was observed in differentiated human neuroblastoma (SH-SY5Y) cells after exposure to 0.25microM of the fungal metabolite gliotoxin for 72h. Further, the involvement of caspases and calpains was demonstrated to underlie the gliotoxin-induced cytotoxic and neurite degenerative effects. The caspase inhibitor Z-VAD-fmk almost completely reduced the neurite degeneration from 40% degeneration of neurites to 5% as compared to control. Inhibition of calpains with calpeptin significantly attenuated gliotoxin-induced cytotoxicity, determined as reduction in total cellular protein content, from 43% to 14% as compared to control cells. Western blot analyses of alphaII-spectrin breakdown fragments confirmed activity of the proteases, and that alphaII-spectrin was cleaved by caspases in gliotoxin-exposed cells. These results show that calpains and caspases have a role in the toxicity of gliotoxin in differentiated SH-SY5Y cells and that the process may be Ca(2+)-mediated.

  • 31.
    Axelsson, Viktoria
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pikkarainen, Katja
    Forsby, Anna
    Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells2006In: Cell Biology and Toxicology, Vol. 22, 127-136 p.Article in journal (Refereed)
  • 32.
    Bajinskis, Ainars
    et al.
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Lindegren, Helene
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Johansson, Lotta
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Low-Dose/Dose-Rate gamma Radiation Depresses Neural Differentiation and Alters Protein Expression Profiles in Neuroblastoma SH-SY5Y Cells and C17.2 Neural Stem Cells2011In: Radiation Research, ISSN 0033-7587, Vol. 175, no 2, 185-192 p.Article in journal (Refereed)
    Abstract [en]

    The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose gamma-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs gamma rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate gamma rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism. (C) 2011 by Radiation Research Society

  • 33. Bal-Price, Anna
    et al.
    Crofton, Kevin M.
    Sachana, Magdalini
    Shafer, Timothy J.
    Behl, Mamta
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hargreaves, Alan
    Landesmann, Brigitte
    Lein, Pamela J.
    Louisse, Jochem
    Monnet-Tschudi, Florianne
    Paini, Alicia
    Rolaki, Alexandra
    Schrattenholz, Andre
    Sunol, Cristina
    van Thriel, Christoph
    Whelan, Maurice
    Fritsche, Ellen
    Putative adverse outcome pathways relevant to neurotoxicity2015In: Critical reviews in toxicology, ISSN 1040-8444, E-ISSN 1547-6898, Vol. 45, no 1, 83-91 p.Article, review/survey (Refereed)
    Abstract [en]

    The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

  • 34. Bavec, Aljosa
    et al.
    Jiang, Yang
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zorko, Matjaz
    Role of cysteine 341 and arginine 348 of GLP-1 receptor in G-protein coupling2007In: Molecular Biology Reports, ISSN 0301-4851, E-ISSN 1573-4978, Vol. 34, no 1, 53-60 p.Article in journal (Refereed)
    Abstract [en]

    We have demonstrated the ability of peptides derived from the third intracellular loop of GLP-1 receptor to differently modulate activity of four different types of G-proteins overexpressed in sf9 cells. In this respect, the involvement of Cys341 in inhibition of Gs and Cys341 in activation of Gs and in inhibition of Gi1, Go, and G11, respectively, indicates their potential role in discrimination between different types of G-proteins. Moreover, these two amino acids from the third intracellular loop might represent an important novel targets for covalent modification by downstream regulators in signaling through GLP-1 receptor.

  • 35.
    Beckman, Marie
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Temporal events in neuronal differentiation and cell death: expression and processing of the Alzheimer's amyloid precursor protein family and a protein at the nuclear pore2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The present study had two major objectives: 1) to elucidate the involvement of Alzheimer’s amyloid precursor protein (APP) family in neuronal differentiation, and the effect of the Alzheimer’s disease (AD)-linked mutation APPV642I on signal transduction; 2) to investigate the fate of the nuclear pore complex protein POM121 during apoptosis and to examine the possibility of using green fluorescent protein (GFP)-labelled POM121 as a non-invasive sensor of apoptosis in living (non-fixed) cells.

    APP is the parent protein of the b-amyloid peptide, which is the major peptide constituent in the “senile plaques” of AD. APP and the homologous amyloid precursor-like proteins, APLP1 and APLP2, are members of the APP family. We compared the temporal expression patterns of these proteins during retinoic acid (RA)-induced neuronal differentiation of human neuroblastoma cells. To this end, we established a quantitative non-radioactive Northern blot assay for APLP1, APLP2 and APP. We found that the transcripts of all three APP family members were increased in response to RA. This occurred simultaneously with progressive neurite outgrowth and increased expression of neuronal markers. In addition, we observed that the increase in APLP2 mRNA was similar to that of APP mRNA, whereas the increase in APLP1 mRNA was significantly higher. The elevated mRNA levels also resulted in an in-creased protein expression of APLP1, APLP2 and the neuronal APP695 isoform. Studies using curcumin (diferuloylmethane), an inhibitor of the transcription factors NFkB/AP-1, and the mitogen-activated protein kinase (MAPK) JNK (c-Jun N-terminal kinase), revealed a diffe-rential regulation of APLP1 and APLP2. Curcumin suppressed the RA-induced mRNA expression of the APP family, in particular that of APLP1. On the protein level, curcumin also reduced the expression of APLP1. In contrast, curcumin induced an accumulation of APLP2, which we propose is due to inhibition of its proteolytic processing. Furthermore, curcumin induced neurite retraction in RA-differentiated cells without affecting their viability. Our results suggest that NFkB/AP-1 signal transduction pathways mediate a co-ordinated regula-tion of the mRNA expression of the APP family and that APLP1 processing is not regulated by the same mechanisms as the processing of APLP2 and APP. Our results are in agreement with important functions for APLP1, APLP2 and APP within the period of neurite extension and synaptic maturation, and a proposed role for these proteins in neuronal differentiation and synaptic plasticity.

    Using a doxycycline-controlled gene expression system (Tet-On), we investigated the effect of wild-type APP695 and the pathogenic familial AD-linked APPV642I mutant on signal transduction. Overexpression was induced at different levels in rat pheochromocytoma (PC12) Tet-On cells. We observed a nerve growth factor-dependent increase in the levels of phosphorylated extracellular regulated kinases 1 and 2 in response to expression of mutant APP. Our results support that increased signalling via MAPKs may have a role in the development of AD. In addition, we found that the inducing agent doxycycline in itself affected cell signalling and protected against oxidative stress. This information is critical for evaluation of the effects of transgene expression using Tet systems.

    Finally, we showed that POM121 is cleaved by a caspase-3-dependent mechanism at aspartate 531 during apoptosis. Characterising the degradation of POM121-GFP in relation to other apoptotic events, revealed that it can be applied as an early non-invasive sensor of nuc-lear apoptosis in living cells using fluorescence microscopy or fluorimetric analysis.

  • 36.
    Beckman, Marie
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kihlmark, Madeleine
    Södertörn University, Stockholm, Sweden.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Nucleus and Nuclear Envelope: Methods for Preparation2010Book (Other academic)
    Abstract [en]

    The cell nucleus of eukaryotic organisms contains the genome surrounded by a nuclear envelope consisting of a double-lipid membrane with embedded nuclear pores and an underlying nuclear lamina. The uniformity in size and density makes it possible to isolate pure intact nuclei at high yields from tissue homogenates by centrifugation through a sucrose cushion. Nuclear envelopes can be prepared from isolated nuclei by enzymatic degradation of their nucleic acid content. The resulting nuclear envelope preparations contain structurally well-conserved inner and outer nuclear membranes with attached ribosomes, nuclear pore complexes and nuclear lamina. Reliable methods for preparation of nuclei and nuclear envelopes play an important role in the successful identification of components that are located in nuclei and in nuclear subcompartments.

  • 37. Bell, Thomas J.
    et al.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eberwine, James
    PAIR technology: exon-specific RNA binding protein isolation in live cells2011In: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2011, 473-486 p.Chapter in book (Refereed)
    Abstract [en]

    RNA-binding proteins (RBPs) are fundamental regulatory proteins for all forms of transcriptional and posttranscriptional control of gene expression. However, isolating RBPs is technically challenging for investigators. Currently, the most widely used techniques to isolate RBPs are in vitro biochemical approaches. Although these approaches have been useful, they have several limitations. One key limitation to using in vitro biochemical approaches is that RBP–RNA interactions are isolated under nonbiological conditions. Here we review a novel experimental approach to identify RBPs called peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology (Zielinski et al., Proc Natl Acad Sci USA 103:1557–1562, 2006). This technology has two significant advantages over traditional approaches. (1) It overcomes the in vitro limitation of biochemical approaches by allowing investigators to isolate RBP–RNA interactions under in vivo conditions. (2) This technology is highly mRNA specific; it isolates RBPs in an exon-specific manner. By selectively targeting alternatively spliced exons with PAIR technology, investigators can isolate splice variant-specific and mRNA region-specific (5-UTR and 3-UTR) RBP complexes for any mRNA of interest.

  • 38.
    Bergqvist, Cecilia
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    The role of nuclear membrane proteins in differentiation and chromatin organization2016Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The nuclear envelope, consisting of an outer and an inner nuclear membrane, surrounds the genomic material. The genomic material (chromatin) is highly structured with (transcriptionally inactive) heterochromatin mostly found in the nuclear periphery and (transcriptionally active) euchromatin mostly found in the nuclear interior. Underlying the nuclear envelope is the nuclear lamina that consists of lamin proteins and nuclear envelope transmembrane proteins (NETs), which organize chromatin in the nuclear periphery. There are several hundred uncharacterized tissue-specific NETs, with only a few linked to cellular differentiation. Induced pluripotent stem cells (iPSCs) enable studies of early differentiation and are a promising tool for cell replacement therapies.

    In this licentiate thesis, we have focused on investigating the role of the inner nuclear membrane protein Samp1 in chromatin organization and cell differentiation. Overexpression of Samp1 induced a fast differentiation of iPSCs, suggesting that Samp1 may be involved in the differentiation process. We have also developed a novel image analysis method to be able to monitor chromatin organization in live cells. Depletion of Samp1 affected chromatin distribution and resulted in increased formation of peripheral heterochromatin, contradictory to what is expected of other characterized NETs. It is possible that Samp1 might have a role in both differentiation and chromatin organization and that future studies might link these two processes together.

  • 39.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Figueroa, Ricardo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Beckman, Marie
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Maxell, Danuta
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Sousa, Paulo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Monitoring of the epigenetic state in live cellsManuscript (preprint) (Other academic)
  • 40.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry. Public Health Agency of Sweden, Sweden.
    Holmström, Petra
    Lindegren, Gunnel
    Lagerqvist, Nina
    Leijon, Mikael
    Falk, Kerstin I.
    Multiplex Nucleic Acid Suspension Bead Arrays for Detection and Subtyping of Filoviruses2015In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 53, no 4, 1368-1370 p.Article in journal (Refereed)
    Abstract [en]

    Here we describe multiplex suspension bead array systems that allow fast and reliable detection of reverse transcriptase (RT) PCR amplified filovirus genomes and also enable subtyping of Ebola virus species and Marburg virus strains. These systems have an analytical sensitivity equivalent to that of RT-PCR.

  • 41.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gudise, Santhosh
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells2017In: Stem Cell Research, ISSN 1873-5061, E-ISSN 1876-7753, Vol. 23, 33-38 p.Article in journal (Refereed)
    Abstract [en]

    The ability of iPSCs (induced pluripotent stem cells) to generate any cell type in the body makes them valuable tools for cell replacement therapies. However, differentiation of iPSCs can be demanding, slowand variable. During differentiation chromatin is re-organized and silent dense heterochromatin becomes tethered to the nuclear periphery by processes involving the nuclear lamina and proteins of the INM(inner nuclearmembrane). The INM protein, Samp1 (Spindle AssociatedMembrane Protein 1) interacts with Lamin A/C and the INMprotein Emerin, which has a chromatin binding LEM(Lap2-Emerin-Man1)-domain. In this paperweinvestigate if Samp1 can play a role in the differentiation of iPSCs. Samp1 levels increased as differentiating iPSCs started to express Lamin A/C. Interestingly, even under pluripotent culturing conditions, ectopic expression of Samp1 induced a rapid differentiation of iPSCs, ofwhich some expressed the neuronal marker beta III-tubulin already after 6 days. This suggests that Samp1 is involved in early differentiation of iPSCs and could potentially be explored as a tool to promote progression of the differentiation process.

  • 42.
    Bergqvist, Cecilia
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jafferali, Mohammed Hakim
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Santosh, Gudise
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Markus, Robert
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Hallberg, Einar
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cellsManuscript (preprint) (Other academic)
  • 43. Bestas, Burcu
    et al.
    Moreno, Pedro M. D.
    Blomberg, K. Emelie M.
    Mohammad, Dara K.
    Saleh, Amer F.
    Sutlu, Tolga
    Nordin, Joel Z.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gustafsson, Manuela O.
    Kharazi, Shabnam
    Piatosa, Barbara
    Roberts, Thomas C.
    Behlke, Mark A.
    Wood, Matthew J. A.
    Gait, Michael J.
    Lundin, Karin E.
    EL Andaloussi, Samir
    Mansson, Robert
    Berglof, Anna
    Wengel, Jesper
    Smith, C. I. Edvard
    Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model2014In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 124, no 9, 4067-4081 p.Article in journal (Refereed)
    Abstract [en]

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

  • 44. Blackshear, Alice
    et al.
    Yamamoto, Mihoko
    Anderson, Brenda J.
    Holmes, Philip V.
    Lundström, Linda
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Robinson, John K.
    Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus2007In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 28, no 5, 1120-1124 p.Article in journal (Refereed)
    Abstract [en]

    The neuropeptide galanin and galanin receptors are widespread throughout cortical, limbic and midbrain areas implicated in reward, learning/memory, pain, drinking and feeding. While many studies have shown that galanin produces a variety of presynaptic and postsynaptic responses, work studying the effects of galanin on neural activation is limited. The present study examined patterns of c-Fos immunoreactivity resulting from intracerebro-ventricular administration of galanin versus saline injection in awake rats. An initial comprehensive qualitative survey was conducted to identify regions of high c-Fos expression followed up with quantitative analysis. Galanin induced a significant increase in c-Fos levels relative to saline-treated controls in dorsomedial hypothalamus and in the central nucleus of the amygdala. This pattern of activation was also produced by galanin receptor type 1 agonist M617. The present findings confirm that galanin upregulates c-Fos activation in hypothalamic nuclei, and supports roles for galanin in central amygdala-mediated food intake, and Pavlovian conditioning.

  • 45. Bocedi, Alessio
    et al.
    Fabrini, Raffaele
    Lo Bello, Mario
    Caccuri, Anna Maria
    Federici, Giorgio
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cornish-Bowden, Athel
    Ricci, Giorgio
    Evolution of Negative Cooperativity in Glutathione Transferase Enabled Preservation of Enzyme Function2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 52, 26739-26749 p.Article in journal (Refereed)
    Abstract [en]

    Negative cooperativity in enzyme reactions, in which the first event makes subsequent events less favorable, is sometimes well understood at the molecular level, but its physiological role has often been obscure. Negative cooperativity occurs in human glutathione transferase (GST) GSTP1-1 when it binds and neutralizes a toxic nitric oxide adduct, the dinitrosyl-diglutathionyl iron complex (DNDGIC). However, the generality of this behavior across the divergent GST family and its evolutionary significance were unclear. To investigate, we studied 16 different GSTs, revealing that negative cooperativity is present only in more recently evolved GSTs, indicating evolutionary drift in this direction. In some variants, Hill coefficients were close to 0.5, the highest degree of negative cooperativity commonly observed (although smaller values of n(H) are theoretically possible). As DNDGIC is also a strong inhibitor of GSTs, we suggest negative cooperativity might have evolved to maintain a residual conjugating activity of GST against toxins even in the presence of high DNDGIC concentrations. Interestingly, two human isoenzymes that play a special protective role, safeguarding DNA from DNDGIC, display a classical half-of-the-sites interaction. Analysis of GST structures identified elements that could play a role in negative cooperativity in GSTs. Beside the well known lock-and-key and clasp motifs, other alternative structural interactions between subunits may be proposed for a few GSTs. Taken together, our findings suggest the evolution of self-preservation of enzyme function as a novel facility emerging from negative cooperativity.

  • 46. Bodor, N
    et al.
    Tóth-Sarudy, E
    Holm, T
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Pallagi, I
    Vass, E
    Buchwald, P
    Langel, U
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Novel, cell-penetrating molecular transporters with flexible backbones and permanently charged side-chains.2007In: J Pharm Pharmacol, ISSN 0022-3573, Vol. 59, no 8, 1065-76 p.Article in journal (Refereed)
  • 47. Brunnström, Åsa
    et al.
    Hamberg, Mats
    Griffiths, William J.
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Claesson, Hans-Erik
    Biosynthesis of 14,15-hepoxilins in human L1236 hodgkin lymphoma cells and eosinophils2011In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 46, no 1, 69-79 p.Article in journal (Refereed)
    Abstract [en]

    Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA(3) 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB(3) 13 (R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-D). Incubation of L1236 cells with synthetic 14,15-HxA(3) 11(S) also led to the formation of 14,15-HxA(3)-C and 14,15-HxA(3)-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA(3) to 14,15-HxA(3)-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.

  • 48. Brunnström, Åsa
    et al.
    Tryselius, Ylva
    Feltenmark, Stina
    Andersson, Erik
    Leksell, Helene
    James, Anna
    Mannervik, Bengt
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Dahlén, Barbro
    Claesson, Hans-Erik
    On the biosynthesis of 15-HETE and eoxin C-4 by human airway epithelial cells2015In: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 121, 83-90 p.Article in journal (Refereed)
    Abstract [en]

    Several lines of evidence indicate that 15-lipoxygenase type 1 (15-LO-1) plays a pathophysiological role in asthma. The aim for this study was to investigate the 15-LO-1 expression and activity in primary human airway epithelial cells cultivated on micro-porous filters at air liquid interface. Incubation of human airway epithelial cells with arachidonic acid led to the formation of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and exposing the cells to bacteria or physical injury markedly increased their production of 15-HETE. The cells were also found to convert arachidonic acid to eoxin C-4 (EXC4). Subcellular fractionation revealed that the conversion of EXA(4) to EXC4 was catalyzed by a soluble glutathione transferase (GST). The GST P1-1 enzyme was found to possess the highest activity of the investigated soluble GSTs. Following IL-4 treatment of airway epithelial cells, microarray analysis confirmed high expression of 15-LO-1 and GST P1-1, and immunohistochemical staining of bronchial biopsies revealed co-localization of 15-LO-1 and GST P1-1 in airway epithelial cells. These results indicate that respiratory infection and cell injury may activate the 15-LO pathway in airway epithelial cells. Furthermore, we also demonstrate that airway epithelial cells have the capacity to produce EXC4.

  • 49.
    Bárány-Wallje, Elsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gaur, Jugnu
    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.
    Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo2007In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 13, 2389-2393 p.Article in journal (Refereed)
    Abstract [en]

    The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55 kDa streptavidin protein was attached to Tp10. When a 5.4 kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

  • 50. Carter, Victoria
    et al.
    Underhill, Ann
    Baber, Ibrahima
    Sylla, Lakamy
    Baby, Mounirou
    Larget-Thiery, Isabelle
    Zettor, Agnès
    Bourgouin, Catherine
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Otvos, Laszlo
    Wade, John D.
    Coulibaly, Mamadou B.
    Traore, Sekou F.
    Tripet, Frederic
    Eggleston, Paul
    Hurd, Hilary
    Killer bee molecules: antimicrobial peptides as effector molecules to target sporogonic stages of Plasmodium2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 11, e1003790Article in journal (Refereed)
    Abstract [en]

    A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

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