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  • 1.
    Araya, Zufan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Tang, Wanjin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hormonal regulation of the human sterol 27-hydroxylase gene CYP27A1.2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 372, no 2, p. 529-534Article in journal (Refereed)
    Abstract [en]

    The mitochondrial sterol 27-hydroxylase (CYP27A1) is a multifunctional cytochrome P450 enzyme that catalyses important hydroxylations in the biosynthesis of bile acids and bioactivation of vitamin D3. Previous results [Babiker, Andersson, Lund, Xiu, Deeb, Reshef, Leitersdorf, Diczfalusy and Bj¨orkhem (1997) J. Biol. Chem. 272, 26253–26261] suggest that CYP27A1 plays an important role in cholesterol homoeostasis and affects atherogenesis. In the present study, the regulation of the human CYP27A1 gene by growth hormone (GH), insulin-like growth factor-1 (IGF-1), dexamethasone, thyroid hormones and PMA was studied. HepG2 cells were transfected transiently with luciferase reporter gene constructs containing DNA fragments flanking the 5_-region of the human CYP27A1 gene. GH, IGF-1 and dexamethasone increased the promoter activity by 2–3-fold, whereas thyroxine (T4) and PMA repressed the activity significantly when measured with luciferase activity expressed in the cells. The endogenous CYP27A1 enzyme activity in the cells was stimulated by GH, IGF-1 and dexamethasone, whereas T4 and PMA inhibited the activity. Experiments with progressive deletion/luciferase reporter gene constructs indicated that the response elements for GH may be localized in a region upstream to position −1094 bp. The putative response elements for dexamethasone were mapped to positions between −792 and −1095 bp. The −451 bp fragment of the human CYP27A1 gene was found to confer the activation by IGF-1, and the inhibition by T4 and PMA. Results of the present study suggest that CYP27A1 is regulated in human cells by hormones and signal-transduction pathways.

  • 2.
    Aspenström, Pontus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Fransson, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Saras, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Rho GTPases have diverse effects on the organization of the actin filament system2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 377, no Pt 2, p. 327-337Article in journal (Refereed)
    Abstract [en]

    The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.

  • 3. Baez, Sofia
    et al.
    Segura-Aguilar, Juan
    Widersten, Mikael
    Johansson, Ann-Sofie
    Mannervik, Bengt
    Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes1997In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 324, p. 25-28Article in journal (Refereed)
  • 4. Barderi, P
    et al.
    Campetella, O
    Frasch, A C
    Santome, JA
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Pettersson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Medical Genetics.
    Cazzulo, JJ
    The NADP+ linked glutamate dehydrogenase from Trypanosoma cruzi: sequence, genomic organization and expression1998In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 330, no 2, p. 951-958Article in journal (Refereed)
    Abstract [en]

    NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.

  • 5. Blombach, Fabian
    et al.
    Launay, Helene
    Snijders, Ambrosius P. L.
    Zorraquino, Violeta
    Wu, Hao
    de Koning, Bart
    Brouns, Stan J. J.
    Ettema, Thijs J. G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Camilloni, Carlo
    Cavalli, Andrea
    Vendruscolo, Michele
    Dickman, Mark J.
    Cabrita, Lisa D.
    LA Teana, Anna
    Benelli, Dario
    Londei, Paola
    Christodoulou, John
    van der Oost, John
    Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 462, p. 373-384Article in journal (Refereed)
    Abstract [en]

    MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix-turn-helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1 ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.

  • 6. Braga, Tiago
    et al.
    Grujic, Mirjana
    Lukinius, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åbrink, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pejler, Gunnar
    Serglycin proteoglycan is required for secretory granule integrity in mucosal mast cells2007In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 403, no 1, p. 49-57Article in journal (Refereed)
    Abstract [en]

    SG (serglycin) PGs (proteoglycans) are strongly implicated in the assembly of MC (mast cell) granules. However, this notion has mainly been on the basis of studies of MCs of the connective tissue subtype, whereas the role of SG PG in mucosal MCs has not been explored. In the present study, we have addressed the latter issue by using mice with an inactivated SG gene. Bone marrow cells were differentiated in vitro into the mucosal MC phenotype, expressing the markers mMCP (mouse MC protease) -1 and -2. Biosynthetic labelling experiments performed on these cells revealed an ~80% reduction of 35SO42− incorporation into PGs recovered from SG−/− cells as compared with SG+/+ counterparts, indicating that SG is the dominating cell-associated PG of mucosal MCs. Moreover, the absence of SG led to defective metachromatic staining of mucosal MCs, both in vivo and in the in vitro-derived mucosal MCs. Ultrastructural analysis showed that granules were present in similar numbers in SG+/+ and SG−/− cells, but that their morphology was markedly affected by the absence of SG, e.g. with electron-dense core formation only seen in SG+/+ granules. Analysis of the MC-specific proteases showed that mMCP-1 and mMCP-7 were completely independent of SG for storage, whereas mMCP-2 showed a partial dependence. In contrast, mMCP-4 and -6, and carboxypeptidase A were strongly dependent on SG for storage. Together, our data indicate that SG PG is of crucial importance for assembly of mature mucosal MC granules, but that the specific dependence on SG for storage varies between individual granule constituents.

  • 7.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Esterbauer, Hermann
    Mannervik, Bengt
    Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases1987In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 247, no 3, p. 707-713Article in journal (Refereed)
    Abstract [en]

    The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

  • 8.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Kinetic independence of the subunits of cytosolic glutathione transferase from the rat1985In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 231, no 2, p. 263-267Article in journal (Refereed)
    Abstract [en]

    The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

  • 9.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 250, no 3, p. 705-711Article in journal (Refereed)
    Abstract [en]

    Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

  • 10.
    Dobritzsch, Doreen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wang, Huaming
    Schneider, Gunter
    Yu, Shukun
    Structural and functional characterization of ochratoxinase, a novel mycotoxin-degrading enzyme2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 462, no 3, p. 441-452Article in journal (Refereed)
    Abstract [en]

    Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 angstrom) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH similar to 6 and 66 degrees C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller beta-sandwich domain. The active site contains an aspartate residue for acid base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.

  • 11.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Catalysis of potato epoxide hydrolase, StEH12005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 390, p. 633-640Article in journal (Refereed)
    Abstract [en]

    The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 mmol·min-1·mg-1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

  • 12. Engel, Stephanie
    et al.
    Scolari, Silvia
    Thaa, Bastian
    Krebs, Nils
    Korte, Thomas
    Herrmann, Andreas
    Veit, Michael
    FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts.2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 425, no 3, p. 567-73Article in journal (Refereed)
    Abstract [en]

    It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA-Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA-Cer with Myr-Pal-YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal-YFP exhibited a much faster mobility compared with HA-Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.

  • 13.
    Eriksson, Jan W
    et al.
    Department of Medicine II, University of Gothenburg, Sahlgren's Hospital, Sweden..
    Lönnroth, P
    Department of Medicine II, University of Gothenburg, Sahlgren's Hospital, Sweden..
    Smith, U
    Department of Medicine II, University of Gothenburg, Sahlgren's Hospital, Sweden..
    Cyclic AMP impairs the rapid effect of insulin to enhance cell-surface insulin-binding capacity in rat adipocytes1992In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 288, no Pt 2, p. 625-629Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to characterize further the interaction between cyclic AMP (cAMP) and insulin binding and action. Rat adipocytes were preincubated at 37 degrees C for 20 min, and after energy depletion with KCN, cell-surface 125I-insulin binding was measured. As recently reported [Eriksson, Lönnroth & Smith (1992) Diabetes 41, 707-714], preincubation with insulin rapidly increased the number of cell-surface insulin binding sites up to approximately 5-fold through recruitment within the plasma membrane. This was completely abolished by the presence of 4 mM-N6-monobutyryl cAMP (a non-hydrolysable cAMP analogue) or 1 microM-isoprenaline, without any apparent change in receptor internalization. Insulin-stimulated receptor tyrosine kinase activity was attenuated by the cAMP analogue only if the exposure of the adipocytes was prolonged to 60 min. The cellular sensitivity to insulin, assessed as 3-O-methylglucose uptake, was markedly decreased by the cAMP analogue, and this could be attributed to the impaired cell-surface binding. However, evidence for post-receptor interactions between cAMP and insulin was also found: an impairment of maximal insulin-stimulated 3-O-methylglucose transport and a delay in the rate of activation of the glucose transport system by insulin. In conclusion, these data demonstrate that beta-adrenergic stimulation and elevated cAMP levels markedly impair the ability of insulin to enhance cell-surface insulin-binding capacity. This novel interaction may be an important mechanism for the cellular insensitivity to insulin produced by cAMP.

  • 14.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Raffalli-Mathieu, Francoise
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 431, no 1, p. 159-167Article in journal (Refereed)
    Abstract [en]

    A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

  • 15. García-Murria, María-Jesús
    et al.
    Karkehabadi, Saeid
    Marín-Navarro, Julia
    Satagopan, Sriram
    Andersson, Inger
    Department of Molecular Biology, Swedish University of Agricultural Sciences.
    Spreitzer, Robert J
    Moreno, Joaquín
    Structural and functional consequences of the replacement of proximal residues Cys(172) and Cys(192) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii.2008In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 411, no 2, p. 241-247Article in journal (Refereed)
    Abstract [en]

    Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

  • 16.
    Gossas, Thomas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation2006In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 398, no 3, p. 393-398Article in journal (Refereed)
    Abstract [en]

    Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the K-D was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and > 100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pK(a) values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate-and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.

  • 17.
    Gralén, Nils
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Philosophy, Mathematics and Science Section.
    The molecular weight of crystalline myogen1939In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 33, no 8, p. 1342-1345Article in journal (Refereed)
  • 18.
    Gralén, Nils
    et al.
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Philosophy, Mathematics and Science Section.
    Svedberg, The
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Philosophy, Mathematics and Science Section.
    Soluble reserve-carbohydrates in the liliifloreae1940In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 34, no 3, p. 234-248Article in journal (Refereed)
  • 19. Haby, Christelle
    et al.
    Larsson, Olof
    Islam, M. Shahidul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Internal Medicine.
    Aunis, Dominique
    Berggren, Per-Olof
    Zwiller, Jean
    Inhibition of serine/threonine protein phosphatases promotes opening of voltage-activated L-type Ca2+ channels in insulin-secreting cells1994In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 298, no Pt 2, p. 341-346Article in journal (Refereed)
    Abstract [en]

    The biological activity of many proteins, including voltage-sensitive ion channels, is controlled by their state of phosphorylation. Ca2+ influx through voltage-activated L-type Ca2+ channels serves as the major stimulatory signal in insulin-secreting cells. We have now investigated the extent to which Ca2+ handling in clonal insulin-secreting RiNm5F cells was affected by okadaic acid, an inhibitor of various serine/threonine protein phosphatases. Whole-cell patch-clamp experiments showed that okadaic acid generated an increase in membrane current, suggesting that it promotes Ca2+ influx through L-type voltage-gated Ca2+ channels probably by modifying their phosphorylation state. Okadaic acid was found to provoke a transient rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i) but had no further effect on the K(+)-induced increase. The Ca2+ transient induced by okadaic acid was dependent on the presence of extracellular Ca2+ and was abolished by D600, a blocker of voltage-activated L-type Ca2+ channels. Concomitant with the rise in [Ca2+]i, okadaic acid induced insulin secretion, a phenomenon that was also dependent on extracellular Ca2+. It is proposed that hyperphosphorylation of voltage-activated L-type Ca2+ channels in insulin-secreting cells lowers the threshold potential for their activation.

  • 20.
    Haitina, Tatjana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 3.
    Klovins, Janis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 3.
    Andersson, Jan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 3.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 3.
    Lagerström, Malin C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 3.
    Larhammar, Dan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 2.
    Larson, Earl T
    Schiöth, Helgi B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. Farmakologi 3.
    Cloning, tissue distribution, pharmacology and three-dimensional modelling of melanocortin receptors 4 and 5 in rainbow trout suggest close evolutionary relationship of these subtypes2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 380, no 2, p. 475-486Article in journal (Refereed)
    Abstract [en]

    The rainbow trout (Oncorhynchus mykiss) is one of the most widely used fish species in aquaculture and physiological research. In the present paper, we report the first cloning, 3D (three-dimensional) modelling, pharmacological characterization and tissue distribution of two melanocortin (MC) receptors in rainbow trout. Phylogenetic analysis indicates that these receptors are orthologues of the human MC4 and MC5 receptors. We created 3D molecular models of these rainbow trout receptors and their human counterparts. These models suggest greater divergence between the two human receptors than between their rainbow trout counterparts. The pharmacological analyses demonstrated that ACTH (adrenocorticotropic hormone) had surprisingly high affinity for the rainbow trout MC4 and MC5 receptors, whereas alpha-, beta- and gamma-MSH (melanocyte-stimulating hormone) had lower affinity. In second-messenger studies, the cyclic MSH analogues MTII and SHU9119 acted as potent agonist and antagonist respectively at the rainbow trout MC4 receptor, indicating that these ligands are suitable for physiological studies in rainbow trout. Interestingly, we found that the rainbow trout MC4 receptor has a natural high-affinity binding site for zinc ions (0.5 microM) indicating that zinc may play an evolutionary conserved role at this receptor. Reverse transcription PCR indicates that the rainbow trout receptors are expressed both in peripheral tissues and in the central nervous system, including the telencephalon, optic tectum and hypothalamus. Overall, this analysis indicates that the rainbow trout MC4 and MC5 receptors have more in common than their mammalian counterparts, which may suggest that these two receptors have a closer evolutionary relationship than the other MC receptor subtypes.

  • 21. Hansson, Lars O.
    et al.
    Widersten, Mikael
    Mannervik, Bengt
    An approach to optimizing the active site in a glutathione transferase by evolution in vitro1999In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 344, p. 93-100Article in journal (Refereed)
  • 22. Hao, Xiao-Yiao
    et al.
    Widersten, Mikael
    Ridderström, Marianne
    Hellman, Ulf
    Mannervik, Bengt
    Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme1994In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 297, p. 59-67Article in journal (Refereed)
  • 23. Hernández-Fisac, Inés
    et al.
    Fernández-Pascual, Sergio
    Ortsäter, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Pizarro-Delgado, Javier
    Martín del Rí­o, Rafael
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Tamarit-Rodriguez, Jorge
    Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets2006In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 400, no 1, p. 81-89Article in journal (Refereed)
    Abstract [en]

    OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernandez-Pascual, Mukala-Nsengu-Tshibangu, Martin del Rio and Tamarit-Rodriguez (2004) Biochem. J. 379,721-729]. In the present study, we have investigated how 10mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-C-14]-Glutamine (at 0.5 and 10.0 mM) conversion to (CO2)-C-14 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 mu M), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucineor glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain a-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

  • 24.
    Islam, M. Shahidul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Internal Medicine.
    Berggren, Per-Olof
    Mobilization of Ca2+ by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone in permeabilized insulin-secreting RINm5F cells: evidence for separate uptake and release compartments in inositol 1,4,5-trisphosphate-sensitive Ca2+ pool1993In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 293, no Pt 2, p. 423-429Article in journal (Refereed)
    Abstract [en]

    We characterized and directly compared the Ca(2+)-releasing actions of two inhibitors of endoplasmic-reticulum (ER) Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ), in electropermeabilized insulin-secreting RINm5F cells. Ambient free calcium concentration ([Ca2+]) was monitored by Ca(2+)-selective mini-electrodes. After ATP-dependent Ca2+ uptake, thapsigargin and tBuBHQ released Ca2+ with and EC50 of approximately 37 nM and approximately 2 microM respectively. Both agents mobilized Ca2+ predominantly from the Ins(1,4,5)P3-sensitive Ca2+ pool, and in this respect thapsigargin was more specific than tBuBHQ. The total increase in [Ca2+] obtained with thapsigargin and Ins(1,4,5)P3 was, on the average, only 7% greater than that with Ins(1,4,5)P3 alone. In contrast, the total increase in [Ca2+] obtained with tBuBHQ and Ins(1,4,5)P3 was 33% greater than that obtained with only InsP3 (P < 0.05). Although Ca2+ was rapidly mobilized by thapsigargin and tBuBHQ, complete depletion of the Ins(1,4,5)P3-sensitive Ca2+ pool was difficult to achieve. After the release by thapsigargin or tBuBHQ, Ins(1,4,5)P3 induced additional Ca2+ release. The additional Ins(1,4,5)P3-induced Ca2+ release was not altered by supramaximal concentrations of thapsigargin and tBuBHQ, or by Bafilomycin A1, an inhibitor of V-type ATPases, but was decreased by prolonged treatment with the ER Ca(2+)-ATPase inhibitors. These results suggest the existence of distinct uptake and release compartments within the Ins(1,4,5)P3-sensitive Ca2+ pool. When treated with the inhibitors, the two compartments became distinguishable on the basis of their Ca2+ permeability. Apparently, thapsigargin and tBuBHQ readily mobilized Ca2+ from the uptake compartment, whereas Ca2+ from the release compartment could be mobilized only very slowly, in the absence of Ins(1,4,5)P3.

  • 25.
    Islam, M. Shahidul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Internal Medicine.
    Kindmark, Henrik
    Larsson, Olof
    Berggren, Per-Olof
    Thiol oxidation by 2,2'-dithiodipyridine causes a reversible increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells: Role for inositol 1,4,5-trisphosphate-sensitive Ca2+ stores1997In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 321, no Pt 2, p. 347-354Article in journal (Refereed)
    Abstract [en]

    2,2'-Dithiodipyridine (2,2'-DTDP), a reactive disulphide that mobilizes Ca2+ from ryanodine-sensitive Ca2+ stores in muscle, induced a biphasic increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells loaded with fura 2. This increase consisted of an early transient followed by a second, slower, rise. The [Ca2+]i transient was dependent on extracellular Ca2+ and disappeared on treatment with nimodipine. The reactive disulphide caused plasma membrane depolarization, as studied by the perforated-patch configuration of the patch-clamp technique. Hence membrane depolarization and opening of the L-type voltage-gated Ca2+ channels were responsible for the first transient in [Ca2+]i. The second slower increase in [Ca2+]i was prolonged but readily reversed by the disulphide-reducing agent 1,4-dithiothreitol. This increase in [Ca2+]i was not decreased by nimodipine or by omission of extracellular Ca2+, but was eliminated when the Ins(1,4,5)P3-sensitive Ca2+ pool was first depleted by carbachol. Ryanodine or its beta-alanyl analogue did not release Ca2+ from intracellular stores, and a high concentration of ryanodine did not inhibit Ca2+ release by 2,2'-DTDP. The disulphide compound suppressed glucose metabolism and decreased the mitochondrial inner-membrane potential. We conclude that thiol oxidation by 2,2'-DTDP affects Ca2+ homeostasis in beta-cells by multiple mechanisms. However, unlike the situation in muscle, in beta-cells 2,2'-DTDP releases Ca2+ from intracellular pools by mechanisms that do not involve activation of ryanodine receptors. Instead, in these cells the Ins(1,4,5)P3-sensitive intracellular Ca2+ store comprises an alternative target for the Ca(2+)-mobilizing action of the reactive disulphide compound.

  • 26.
    Islam, M. Shahidul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Internal Medicine.
    Larsson, O.
    Nilsson, T.
    Berggren, Per-Olof
    Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic beta-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor1995In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 306, no Pt 3, p. 679-686Article in journal (Refereed)
    Abstract [en]

    In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+]i. We studied the effects of caffeine on [Ca2+]i by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+]i. The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+]i. Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+]i in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+]i by causing closure of ATP-sensitive K+ channels (KATP channels). Caffeine also caused inhibition of KATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+]i and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine (> 10 mM) lowered [Ca2+]i. This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+]i, induced fast oscillations in [Ca2+]i. The latter effect was likely to be attributable to the cyclic AMP-elevating action of caffeine, leading to phosphorylation of voltage-gated Ca2+ channels. Hence, in beta-cells, caffeine-induced changes in [Ca2+]i are not due to any interaction with intracellular Ca2+ pools. In these cells, a direct interference with KATP channel- and L-type voltage-gated Ca(2+)-channel activity is the underlying mechanism by which caffeine increases or decreases [Ca2+]i.

  • 27. Jacobson, A
    et al.
    Brinck, J
    Briskin, M J
    Spicer, A P
    Heldin, P
    Expression of human hyaluronan synthases in response to external stimuli.2000In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 348 Pt 1, p. 29-35Article in journal (Refereed)
    Abstract [en]

    In the present study we have investigated the expression of mRNAs for hyaluronan synthase isoforms (HAS1, HAS2 and HAS3) in different cells in response to various stimuli. Human mesothelial cells, which synthesize large amounts of hyaluronan, express mRNAs encoding all three HAS isoforms, whereas their transformed counterparts, mesothelioma cells, which produce only minute amounts of hyaluronan, express only HAS3 mRNA. Human lung fibroblasts and the glioma cell line U-118 MG express only the HAS2 and HAS3 genes. The expression of the transcripts was higher in subconfluent than in confluent cultures and was well correlated with the production of hyaluronan by the cells. Stimulation of mesothelial cells with platelet-derived growth factor-BB induced an up-regulation of mRNA for HAS2 to a maximum after 6 h of stimulation; HAS1 and HAS3 genes were only induced slightly. Transforming growth factor-beta1 reduced HAS2 mRNA slightly, and hydrocortisone reduced it strongly, within 6 h of stimulation in mesothelial cell cultures but did not significantly affect the expression of mRNAs for HAS1 and HAS3. Induction of HAS1 and HAS2 protein levels in response to the stimuli above correlated with HAS transcript levels. Thus the expression of the three HAS isoforms is more prominent in growing cells than in resting cells and is differentially regulated by various stimuli suggesting distinct functional roles of the three proteins.

  • 28. Kass, G E
    et al.
    Webb, D L
    Karolinska inst, Stockholm, Sweden.
    Chow, S C
    Llopis, J
    Berggren, P O
    Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt1994In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 302, p. 5-9Article in journal (Refereed)
    Abstract [en]

    In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.

  • 29.
    Koch, Sina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Tugues, Sonia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Li, Xiujuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Gualandi, Laura
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Signal transduction by vascular endothelial growth factor receptors2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 437, p. 169-183Article, review/survey (Refereed)
    Abstract [en]

    VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.

  • 30.
    Kolm, Rüdiger H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Zhang, Y.uesheng
    Talalay, Paul
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Isothiocyanates as substrates for human glutathione transferases: structure-activity studies1995In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 311, p. 453-459Article in journal (Refereed)
    Abstract [en]

    The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.

  • 31.
    Larsson, Mårten
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjälm, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sakwe, Amos M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Höglund, Anna-Stina
    Larsson, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Robinson, Robert C
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sundberg, Christian
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rask, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 373, no 2, p. 381-391Article in journal (Refereed)
    Abstract [en]

    Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

  • 32.
    Li, Lingli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Asteriou, Trias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Bernert, Berit
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Paraskevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB2007In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 404, p. 327-336Article in journal (Refereed)
    Abstract [en]

    The glycosaminoglycan hyaluronan is important in many tissue-repair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK 1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-kappa B (nuclear factor KB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-beta 1 (transforming growth factor-beta 1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Imponantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hennes-1, inhibited PDGF-BB-stimulated [H-3]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.

  • 33.
    Li, Shi-Sheng
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Gullbo, Joachim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Lindholm, Petra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Thunberg, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Samuelsson, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Bohlin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Claeson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Ligatoxin B, a new cytotoxic protein with a novel helix-turn-helix DNA-binding domain from the mistletoe Phoradendron liga2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 366, no Pt 2, p. 405-13Article in journal (Refereed)
    Abstract [en]

    A new basic protein, designated ligatoxin B, containing 46 amino acid residues has been isolated from the mistletoe Phoradendron liga (Gill.) Eichl. (Viscaceae). The protein's primary structure, determined unambiguously using a combination of automated Edman degradation, trypsin enzymic digestion, and tandem MS analysis, was 1-KSCCPSTTAR-NIYNTCRLTG-ASRSVCASLS-GCKIISGSTC-DSGWNH-46. Ligatoxin B exhibited in vitro cytotoxic activities on the human lymphoma cell line U-937-GTB and the primary multidrug-resistant renal adenocarcinoma cell line ACHN, with IC50 values of 1.8 microM and 3.2 microM respectively. Sequence alignment with other thionins identified a new member of the class 3 thionins, ligatoxin B, which is similar to the earlier described ligatoxin A. As predicted by the method of homology modelling, ligatoxin B shares a three-dimensional structure with the viscotoxins and purothionins and so may have the same mode of cytotoxic action. The novel similarities observed by structural comparison of the helix-turn-helix (HTH) motifs of the thionins, including ligatoxin B, and the HTH DNA-binding proteins, led us to propose the working hypothesis that thionins represent a new group of DNA-binding proteins. This working hypothesis could be useful in further dissecting the molecular mechanisms of thionin cytotoxicity and of thionin opposition to multidrug resistance, and useful in clarifying the physiological function of thionins in plants.

  • 34.
    Lidholt, Kerstin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Erikssson, I
    Kjellen, L
    Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate.1995In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 311, no 1, p. 233-238Article in journal (Refereed)
    Abstract [en]

    Proteoglycans (PGs), biosynthetically labelled with [S-35]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the S-35-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [S-35]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these S-35-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [S-35]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) similar to 30000) than the CS chains in the NA-PGs (M(r) similar to 25000). CS chains in the hybrid PGs were slightly smaller (M(r) similar to 20000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20 %) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [S-35]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.

  • 35.
    Lidholt, Kerstin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Riesenfeld, J
    Jacobsson, K G
    Feingold, D S
    Lindahl, U
    Biosynthesis of heparin: Modulation of polysaccharide chain length in a cell-free system1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 254, no 2, p. 571-578Article in journal (Refereed)
    Abstract [en]

    The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.

  • 36. Lorenzo, Carmen
    et al.
    Salinas, Gustavo
    Brugnini, Andreina
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    González-Sapienza, Gualberto
    Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 369, no Pt 1, p. 191-198Article in journal (Refereed)
    Abstract [en]

    Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.

  • 37. Lövkvist Wallström, Eva
    et al.
    Takao, Koichi
    Wendt, Anna
    Vargiu, Cristina
    Yin, Hong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Persson, Lo
    Importance of the 3' untranslated region of ornithine decarboxylase mRNA in the translational regulation of the enzyme2001In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 356, no Pt 2, p. 627-34Article in journal (Refereed)
    Abstract [en]

    Translational regulation of ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of polyamines, appears to be an important mechanism in the strong feedback control as well as in the hypotonic induction of the enzyme. However, the exact mechanisms are not yet understood. The ODC mRNA has long 5' and 3' untranslated regions (UTRs) which may be involved in the translational control of the enzyme. In the present study we have used a series of stable transfectants of Chinese Hamster ovary cells expressing ODC mRNAs with various truncations in the 5' and 3' UTRs to investigate the importance of these regions. It is demonstrated that neither the 5' UTR nor the 3' UTR appears to be involved in the polyamine-mediated feedback control of ODC synthesis. The hypotonic induction of ODC, on the other hand, was shown to be highly dependent on the presence of the 3' UTR, but not on the 5' UTR, of ODC mRNA. Cells expressing ODC mRNAs lacking the 3' UTR showed no, or only a very slight, induction of ODC whether the 5' UTR was present or not, whereas the cell lines expressing ODC mRNAs containing the 3' UTR (with or without the 5' UTR) markedly induced ODC after a hypotonic shock. The present finding of a role for the ODC mRNA 3' UTR in the hypotonic induction of ODC is the first demonstration of a specific effect of the 3' UTR in the regulation of ODC.

  • 38. Mannervik, Bengt
    et al.
    Awasthi, Yogesh C.
    Board, Philip G.
    Hayes, John D.
    DiIlio, Carmine
    Ketterer, Brian
    Listowsky, Irving
    Morgenstern, Ralf
    Muramatsu, Masami
    Pearson, William R.
    Pickett, Cecil B.
    Sato, K.
    Widersten, Mikael
    Wolf, C. Roland
    NOMENCLATURE FOR HUMAN GLUTATHIONE TRANSFERASES1992In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 282, p. 305-306Article in journal (Refereed)
  • 39.
    Maurer, Dirk
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lohkamp, Bernhard
    Krumpel, Michael
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism2018In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 475, no 14, p. 2395-2416Article in journal (Refereed)
    Abstract [en]

    β-Ureidopropionase (βUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like βUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human βUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N-carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer–dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly βUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer–dimer interface, effectively positioning the catalytically important Glu207 in the active site.

  • 40.
    Metzendorf, Christoph
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Wu, Wenlin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Lind, Maria I
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
    Overexpression of Drosophila mitoferrin in l(2)mbn cells results in dysregulation of Fer1HCH expression2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 421, no Part 3, p. 463-471Article in journal (Refereed)
    Abstract [en]

    Mrs3p and Mrs4p (Mrs3/4p) are yeast mitochondrial iron carrier proteins that play important roles in ISC (iron-sulphur cluster) and haem biosynthesis. At low iron conditions, mitochondrial and cytoplasmic ISC protein maturation is correlated with MRS3/4 expression. Zebrafish mitoferrin1 (mfrn1), one of two MRS3/4 orthologues, is essential for erythropoiesis, but little is known about the ubiquitously expressed paralogue mfrn2. In the present study we identified a single mitoferrin gene (dmfrn) in the genome of Drosophila melanogaster, which is probably an orthologue of mfrn2. Overexpression of dmfrn in the Drosophila l(2)mbn cell line (mbn-dmfrn) resulted in decreased binding between IRP-1A (iron regulatory protein 1A) and stem-loop RNA structures referred to as IREs (iron responsive elements). mbn-dmfrn cell lines also had increased cytoplasmic aconitase activity and slightly decreased iron content. In contrast, iron loading results in decreased IRP-1A-IRE binding, but increased cellular iron content, in experimental mbn-dmfrn and control cell lines. Iron loading also increases cytoplasmic aconitase activity in all cell lines, but with slightly higher activity observed in mbn-dmfrn cells. From this we concluded that dmfrn overexpression stimulates cytoplasmic ISC protein maturation, as has been reported for MRS3/4 overexpression. Compared with control cell lines, mbn-dmfrn cells had higher Fer1HCH (ferritin 1 heavy chain homologue) transcript and protein levels. RNA interference of the putative Drosophila orthologue of human ABCB7, a mitochondrial transporter involved in cytoplasmic ISC protein maturation, restored Fer1HCH transcript levels of iron-treated mbn-dmfrn cells to those of control cells grown in normal medium. These results suggest that dmfrn overexpression in l(2)mbn cells causes an 'overestimation' of the cellular iron content, and that regulation of Fer1HCH transcript abundance probably depends on cytoplasmic ISC protein maturation.

  • 41.
    Mystkowski, Edmund Marcel
    Uppsala University, Disciplinary Domain of Humanities and Social Sciences, Faculty of Philosophy, Mathematics and Science Section.
    Ultracentrifugal studies of compounds of proteins with polysaccharides: Compounds between proteins and glycogen1937In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 31, no 5, p. 716-720Article in journal (Refereed)
  • 42. Neuschäfer-Rube, Frank
    et al.
    Hermosilla, Ricardo
    Rehwald, Mathias
    Rönnstrand, Lars
    Schülein, Ralf
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Püschel, Gerhard Paul
    Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 379, no Pt 3, p. 573-585Article in journal (Refereed)
    Abstract [en]

    hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.

  • 43.
    Nilsson, B
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Grossberger, D
    Nilsson Ekdahl, K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Riegert, P
    Becherer, D J
    Nilsson, U R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Lambris, J D
    Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.1992In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 282 ( Pt 3), p. 715-721Article in journal (Refereed)
    Abstract [en]

    In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

  • 44.
    Nilsson, B
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Nilsson Ekdahl, K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Avila, D
    Nilsson, U R
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Lambris, J D
    Neoantigens in complement component C3 as detected by monoclonal antibodies. Mapping of the recognized epitopes by synthetic peptides.1990In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 268, no 1, p. 55-61Article in journal (Refereed)
    Abstract [en]

    The different fragments of the third complement component, C3, generated upon complement activation/inactivation have the ability to bind to several other complement components and receptors as well as to proteins of foreign origin. These multiple reactivities of C3 fragments are associated with a series of conformational changes occurring in the C3 molecule during its degradation. The conformations acquired by the different C3 fragments are also associated with the exposure of neoantigenic epitopes that are specific for (a) particular fragment(s). In order to study these epitopes and thus the conformational changes occurring in C3, monoclonal antibodies (mAbs) recognizing such epitopes were produced in Balb/c mice after immunization with denatured human C3. Two of the three antibodies (7D84.1 and 7D264.6) presented in this study recognized predominantly surface-bound iC3b, and one mAb (7D323.1) recognized both surface-bound and fluid-phase iC3b. Although none of the mAbs recognized any other fluid-phase C3 fragment, all three antibodies detected micro-titre-plate-fixed C3b and iC3b, but not C3c or C3d. In addition to the reaction with human C3, mAb 7D323.1 also bound to micro-titre-plate-fixed rabbit C3. The epitopes recognized by the three mAbs were further localized by using synthetic peptides that were designed on the basis of the differential binding of the mAbs to the C3 fragments. All three antibodies reacted with C3-(924-965)-peptide, which represents the region of C3 between the kallikrein-cleavage site (923-924) and the elastase-cleavage site (965-966). On the basis of the binding of the mAbs to five different overlapping peptides spanning the region between residues 924 and 965 of the human C3 sequence, and the sequence similarity between human C3 and rabbit C3 within this area, the epitopes recognized by these antibodies are mapped. The contribution of the individual amino acid residues in the formation of the epitopes is discussed.

  • 45.
    Niyomrattanakit, Pornwaratt
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Yahorava, Sviatlana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Mutule, Ilze
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Mutulis, Felikss
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Petrovska, Ramona
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Prusis, Peteris
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Katzenmeier, Gerd
    Wikberg, Jarl E S
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Pharmacology.
    Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides2006In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 397, no 1, p. 203-211Article in journal (Refereed)
    Abstract [en]

    The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.

  • 46. Politz, Oliver
    et al.
    Gratchev, Alexei
    McCourt, Peter A. G.
    Schledzewski, Kai
    Guillot, Pierre
    Johansson, Sophie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Svineng, Gunborg
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Franke, Peter
    Kannicht, Christoph
    Kzhyshkowska, Julia
    Longati, Paola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Velten, Florian W.
    Johansson, Staffan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Goerdt, Sergij
    Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, p. 155-164Article in journal (Refereed)
  • 47. Poon, Ivan K H
    et al.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hulett, Mark D
    Parish, Christopher R
    Regulation of histidine-rich glycoprotein (HRG) function via plasmin-mediated proteolytic cleavage2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 424, no Part 1, p. 27-37Article in journal (Refereed)
    Abstract [en]

    The plasminogen/plasmin system is involved in a variety of normal physiological and pathological processes, including tissue remodelling, angiogenesis and tumour metastasis. Plasminogen activators and receptors for plasminogen/plasminogen activators are essential for the processing of plasminogen to form the active serine protease plasmin. Plasmin can in turn positively or negatively regulate further plasminogen activation via plasmin-mediated cleavage of receptors and activators. HRG (histidine-rich glycoprotein), a relatively abundant (approx. 100-150 microg/ml) plasma glycoprotein, has a multi-domain structure that can interact with many ligands, including Zn2+, heparin, HS (heparan sulfate) and plasminogen. HRG has been shown to function as an adaptor molecule to tether plasminogen to GAG (glycosaminoglycan)-bearing surfaces and to regulate plasminogen activation via various mechanisms. As HRG itself is sensitive to plasmin cleavage, the present study examines in detail the cleavage of human HRG by plasmin and the effect of this cleavage on various functions of HRG. HRG fragments, generated by plasmin cleavage, are held together by disulfide linkages and are not released from the molecule under non-reducing conditions. Plasmin-mediated cleavage partially inhibited HRG binding to cell surface HS, but enhanced HRG binding to necrotic cells and to plasminogen. However, both intact and plasmin-cleaved HRG enhanced the binding of plasminogen to heparin-coated surfaces to a similar extent. Furthermore, the presence of heparin, Zn2+ or acidic pH was found to protect HRG from plasmin cleavage. Thus proteolytic cleavage of HRG by plasmin may provide a feedback mechanism to regulate the effects of HRG on the plasminogen/plasmin system and other functions of HRG.

  • 48. Raffalli-Mathieu, Francoise
    et al.
    Carolina, Orre
    Stridsberg, Mats
    Hansson Edalat, Maryam
    Mannervik, Bengt
    Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells2008In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 414, no 1, p. 103-109Article in journal (Refereed)
  • 49.
    Raffalli-Mathieu, Françoise
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Orre, Carolina
    Stridsberg, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Hansson Edalat, Maryam
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells2008In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 414, no 1, p. 103-109Article in journal (Refereed)
    Abstract [en]

    hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Delta(5)-Delta(4) double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3beta-hydroxysteroids catalysed by 3beta-HSD (3beta-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3beta-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Delta(5)-Delta(4) isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Delta(5)-androstene-3,17-dione into Delta(4)-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3beta-hydroxysteroid oxidoreductase activity of 3beta-HSD. The results indicate that the lower isomerase activity of 3beta-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.

  • 50.
    Reyhani, Vahid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Seddigh, Pegah
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Guss, Bengt
    Gustafsson, Renata
    Rask, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Rubin, Kristofer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fibrin binds to collagen and provides a bridge for alpha V beta 3 integrin-dependent contraction of collagen gels2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 462, no P1, p. 113-123Article in journal (Refereed)
    Abstract [en]

    The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through alpha V beta 3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via alpha V beta 3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects alpha V beta 3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

12 1 - 50 of 67
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