| 1. |
- Eriksson, Hans, et al.
(författare)
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Evidence for the key role of the adipocyte cGMP-inhibited cAMP phosphodiesterase in the antilipolytic action of insulin
- 1995
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002. ; 1266:1, s. 101-107
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Tidskriftsartikel (refereegranskat)abstract
- Enhancement of cAMP degradation by increased cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) activity is thought to be an important component of the mechanism whereby insulin counteracts catecholamine-induced lipolysis in adipocytes. In this study the selective cGI-PDE inhibitor OPC3911 was used to evaluate this role of cGI-PDE activation in intact rat adipocytes with special reference to changes in cAMP levels measured as cAMP-dependent protein kinase (cAMP-PK) activity ratios. OPC3911 completely blocked (IC50 = 0.3 microM) the maximal inhibitory effect of insulin on noradrenaline-induced lipolysis and the net dephosphorylation of hormone-sensitive lipase and other intracellular target proteins for insulin action, whereas insulin-induced lipogenesis was not changed. The effect of OPC3911 on cAMP-PK activity ratios at different levels of lipolysis achieved by noradrenaline stimulation revealed that the reduction of cAMP-PK caused by 1 nM insulin was completely blocked by 3 microM OPC3911. The effect of OPC3911 was not due to an excessive increase in cellular cAMP resulting in 'supramaximal' lipolysis unresponsive to insulin. These data demonstrate that reduction in cAMP levels by the activation of cGI-PDE may be sufficient to account for the antilipolytic action of insulin.
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| 2. |
- Andersson, Eva, et al.
(författare)
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The metabolism of vitamin A to 3,4-didehydroretinol can be demonstrated in human keratinocytes, melanoma cells and HeLa cells, and is correlated to cellular retinoid-binding protein expression
- 1994
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1224:3, s. 349-354
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Tidskriftsartikel (refereegranskat)abstract
- Conversion of retinol to 3,4-didehydroretinol is probably a rate-limiting step in the formation of 3,4-didehydroretinoic acid, a candidate ligand for nuclear retinoid receptors in human epidermal keratinocytes. To investigate whether this metabolic pathway also exists in other cell systems, we compared the retinoid concentrations and the bioconversion of [3H]retinol to [3H]3,4-didehydroretinol in human primary keratinocytes, human cervical carcinoma (HeLa) cells, human melanoma (JKM86-4) cells, monkey kidney epithelium (CV-1) cells, and murine teratocarcinoma (F9) cells. The cellular retinol concentration ranged from 2.33 to 99.1 pmol/mg protein with the highest values observed in keratinocytes. 3,4-Didehydroretinol was only detected in cells of human origin and its concentration ranged from 0.24 pmol/mg in HeLa to 34.6 pmol/mg in the keratinocytes. Incubation with [3H]retinol for 1–24 h resulted in a rapid appearance of [3H]3,4-didehydroretinol in human keratinocytes, and to a lesser extent in HeLa and melanoma cells, but not in the other cells. Analysis of cellular retinol- and retinoic acid-binding protein concentrations showed a correlation to the cells' ability to accumulate 3,4-didehydroretinol, suggesting a role for these proteins in the 3,4-didehydro metabolic pathway. The combined results suggest that although 3,4-didehydroretinol is most typical for human keratinocytes, studies of its metabolism are also feasible in HeLa cells which contain low levels of retinoid-binding proteins.
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| 3. |
- Emanuelsson, Olof, et al.
(författare)
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Prediction of organellar targeting signals.
- 2001
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 1541:1-2, s. 114-119
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Tidskriftsartikel (refereegranskat)abstract
- The subcellular location of a protein is an important characteristic with functional implications, and hence the problem of predicting subcellular localization from the amino acid sequence has received a fair amount of attention from the bioinformatics community. This review attempts to summarize the present state of the art in the field.
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| 4. |
- Eriksson, S, et al.
(författare)
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Matrix association of early- and late-replicating chromatin studied by single-cell electrophoresis
- 2002
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 1590:1-3, s. 103-108
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Tidskriftsartikel (refereegranskat)abstract
- CHO-K1 cells were synchronized at the G(1)/S border by mitotic shake-off and aphidicolin incubation. Pulse-labeling with tritium was done at 30 min, 2 or 5 h into the S-phase, with chase incubations for different times in non-radioactive medium. The cells were subjected to neutral microelectrophoresis to extend the DNA into "comets," after which the label was visualized through autoradiography. At zero chase time, all label was positioned in the head. The displacement of label into the tails increased with time, reaching a maximum at about 5 h after the pulse. A lag phase of 2 - 3 It was observed for the early-labeled cells before the displacement started. Also, more label was released after overnight serum starvation, but this was reversed through a 3-h incubation at normal growth conditions. It was found that late-replicating chromatin is organized in larger domains than early-replicating chromatin, and DNA polymerase seems to be an important organizer. Early-replicating chromatin has other important attachments to the nuclear matrix, dependent on metabolic activity.
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| 5. |
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| 6. |
- Helander, Anders, et al.
(författare)
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Mechanisms for Plasma‑Mediated Activation of Human Blood Cell Aldehyde Dehydrogenase
- 1992
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 1136:3, s. 259-264
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Tidskriftsartikel (refereegranskat)abstract
- Aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activity assays were carried out on isolated human blood cells in phosphate-buffered saline (PBS) and in PBS mixed with human plasma. In assays with intact erythrocytes or sonicated leukocytes, the presence of 50% (v/v) or greater of plasma in the reaction mixtures produced a 2-fold increase in the rate of aldehyde oxidation. In corresponding assays with sonicated erythrocyte samples, the ALDH activity was enhanced on an average 1.5-fold, whereas a slight decrease was observed in assays with intact leukocytes. The ALDH inhibitor disulfiram almost completely abolished the enzyme activity both in the absence and presence of plasma. In assays with sonicated leukocytes, the activation effect could be antagonized by EDTA, indicating that it was caused largely by divalent cations. With sonicated erythrocytes, a significantly reduced ALDH activity was found only with the highest concentration of EDTA tested, and since a similar reduction was obtained also when plasma was omitted, the plasma-mediated activation of erythrocyte ALDH was suggested to be due to a different mechanism. After separation of plasma by gel filtration, an active fraction was identified by GC-MS and 1H-NMR to contain pyruvic acid, lactic acid and glucose. When tested at physiological plasma concentrations, pyruvic acid caused an increase in erythrocyte ALDH activity similar to that obtained with plasma, while lactic acid and glucose did not. Pyruvic acid did not activate the leukocyte ALDH. Based on these results, it is indicated that the plasma-mediated activation of erythrocyte ALDH is due to pyruvic acid, which reoxidizes NADH via lactate dehydrogenase (EC 1.1.1.27) and, thereby, increases the rate of dissociation of NADH from the terminal enzyme-NADH complex, the rate-limiting step in the ALDH pathway.
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| 7. |
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| 8. |
- Kass, G E, et al.
(författare)
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Two separate plasma membrane Ca2+ carriers participate in receptor-mediated Ca2+ influx in rat hepatocytes.
- 1994
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1223:2, s. 226-33
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane Ca2+ carrier system involved in receptor-mediated Ca2+ entry was studied. Using the Ca2+ readdition protocol, the rate of cytosolic free Ca2+ concentration ([Ca2+]i) increase in vasopressin-pretreated hepatocytes was significantly higher than in thapsigargin- or 2,5-di(tert-butyl)hydroquinone-pretreated cells. The addition of Mn2+ to unstimulated hepatocytes resulted in a biphasic quench of fura-2 fluorescence. After an initial phase that was fast in rate but of short duration, the rate of fura-2 quench by Mn2+ became much slower and lasted until all the cellular fura-2 was quenched. Pretreatment of the cells with vasopressin only accelerated the rate of the latter phase but not of the initial one. In agonist-stimulated cells, acidification of the extracellular medium or the presence of ruthenium red, econazole or SK&F 96365 decreased the rates of both [Ca2+]i increase and Mn2+ entry upon addition of the respective cation. By contrast, neomycin and N-tosyl-L-phenylalanine chloromethyl ketone markedly decreased the rate of [Ca2+]i increase upon Ca2+ readdition but had no effect on vasopressin-stimulated Mn2+ entry. None of the treatments affected the ability of vasopressin and thapsigargin to mobilize the internal Ca2+ store. It is concluded that in hepatocytes the two pathways of receptor-mediated Ca2+ entry control two distinct yet pharmacologically related cation carriers.
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| 9. |
- Lindmark, Maria, 1972-, et al.
(författare)
-
Synaptotagmin II could confer Ca(2+) sensitivity to phagocytosis in human neutrophils.
- 2002
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Ingår i: Biochimica et biophysica acta. - 0006-3002. ; 1590:1-3, s. 159-166
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Tidskriftsartikel (refereegranskat)abstract
- Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.
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| 10. |
- Lundqvist, Helen, et al.
(författare)
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Neutrophil control of formylmethionyl-leucyl-phenylalanine induced mobilization of secretory vesicles and NADPH-oxidase activation: Effect of an association of the ligand-receptor complex to the cytoskeleton
- 1994
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1224:1, s. 43-50
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Tidskriftsartikel (refereegranskat)abstract
- The stimulus formylmethionyl-leucyl-phenylalanine (FMLP) interacts with neutrophils and generates signal(s) in the cells that induces mobilization of the secretory vesicles as well as activation of the superoxide anion/hydrogen peroxide generating NADPH-oxidase. Binding, at 15°C, of FMLP to its neutrophil surface receptor is followed by an association of the ligand-receptor complex to the cell cytoskeleton, and this association occurs concomitant with a desensitization of the cells with respect to activation of the NADPH-oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself, but rather that the association of the ligand-receptor complex to the cytoskeleton eliminates the capacity of the complex to generate the signal(s) that activates the NADPH-oxidase. The cytoskeleton associated ligand-receptor complex generates, however, the signal(s) responsible for mobilization of the secretory vesicles, to the plasma membrane, and this mobilization occurs without any increase in the intracellular concentration of free Ca2+.
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| 11. |
- Löfgren, Ragnhild, et al.
(författare)
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CR3, FcγRIIA and FcγRIIIB induce activation of the respiratory burst in human neutrophils : the role of intracellular Ca2+, phospholipase D and tyrosine phosphorylation
- 1999
-
Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 1452:1, s. 46-59
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Tidskriftsartikel (refereegranskat)abstract
- Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcγRIIA and FcγRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcγRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcγRIIA Pansorbins induced an even weaker signal. However, FcγRIIA induced strong phosphorylation of p72syk whereas FcγRIIIB induced only a very weak p72syk phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72syk was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72syk that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72syk phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcγRIIA and FcγRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72syk is an early signal in the cascade induced by FcγRIIA but not by CR3.
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| 12. |
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| 13. |
- Nilsson Ekdahl, Kristina, et al.
(författare)
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Effects of epinephrine, glucagon and insulin on the activity and degree of phosphorylation of fructose-1,6-bisphosphatase in cultured hepatocytes.
- 1987
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 929:3, s. 318-326
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Tidskriftsartikel (refereegranskat)abstract
- The effects of epinephrine, glucagon and insulin on the activity and degree of phosphorylation of fructose-1,6-bisphosphatase in isolated hepatocytes maintained in cell culture for 24 h were investigated. Epinephrine caused a rapid decrease in the apparent Km monitored as the activity ratio between the activity at 12.5 and 83 microM fructose-1,6-bisphosphate, reaching a maximum after 5 min. Glucagon caused a slower and less pronounced activation, and insulin caused an equally slow increase in Km. The effect of epinephrine and glucagon was completely reciprocated by insulin and the action of insulin was totally erased by the other two. Glucagon stimulated the incorporation of [32P]phosphate into fructose-1,6-bisphosphatase from about 2.5 to 4.2 mol/mol enzyme and epinephrine to 3.5 mol/mol. The effect of the two hormones acting together was cumulative. Insulin brought about a decrease in the degree of phosphorylation to 2.0 mol/mol. The effect of epinephrine was shown to be caused by the beta-receptors, since it was completely blocked by propanolol (a beta-antagonist) and remained unaffected by the presence of phentolamine (an alpha-antagonist).
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| 14. |
- Nånberg, Eewa, 1957-, et al.
(författare)
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Alpha-1-adrenergic inositol trisphosphate production in brown adipocytes is Na+ dependent
- 1987
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 930:3, s. 438-445
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Tidskriftsartikel (refereegranskat)abstract
- In order to investigate the ionic requirements for inositol trisphosphate production, brown adipocytes were prelabelled with myo-[3H]inositol and the formation of inositol trisphosphates and inositol bisphosphates as a consequence of alpha 1-adrenergic stimulation was monitored. Omission of Ca2+ from the incubation medium diminished the norepinephrine-induced increase in inositol trisphosphate levels, but it would seem that this reduction can be fully accounted for by a decreased level of the 'inactive' isomer inositol 1,3,4-trisphosphate. Omission of Na+ fully abolished the norepinephrine-induced inositol trisphosphate response. However, it was observed that the presence of Li+ in the incubation medium could fully reconstitute the ability of the cells to yield the early response of inositol trisphosphate production; Li+ could, however, not substitute for Na+ in the entire alpha 1-adrenergic cellular pathway. It was concluded that the Na+-dependent step is found in the coupling mechanism between the alpha 1-receptor and the activation of the phosphodiesterase responsible for inositol trisphosphate production. Thus, all events in the alpha 1-adrenergic pathway which are consequences of IP3 production should appear to be Na+-dependent in these cells.
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| 15. |
- Nånberg, Eewa, 1957-, et al.
(författare)
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Alpha-adrenergic effects on 86Rb+(K+) potentials and fluxes in brown fat cells
- 1984
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 804:3, s. 291-300
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Tidskriftsartikel (refereegranskat)abstract
- Net K+ fluxes in isolated hamster brown fat cells were studied by the use of the K+ analogue 86Rb+. In isolated cells, cold-stored overnight to diminish K+ gradients, an equilibrium 86Rb+ (K+) clearance value of 27 microliter/million cells was obtained after 30 min incubation at 37 degrees C. This corresponds to a 10-fold K+ gradient over the plasma membrane, and a K+ potential of about -60 mV. The attainment of this equilibrium was dependent upon the presence of Na+ in the extracellular medium, and the uptake was fully inhibited by the (Na+ + K+)-ATPase inhibitor ouabain. Ouabain had, however, no significant acute effect on the maximal rate of thermogenesis achieved after norepinephrine stimulation of the cells, but if the restoration of ionic equilibrium was inhibited by ouabain in prolonged incubations, a decreased thermogenesis was observed. This was probably due to the low cytosolic K+ content then encountered, and the resulting inhibition of lipolysis. The addition of norepinephrine to cells in which 86Rb+ (K+) equilibrium had been attained resulted in a rapid efflux of 86Rb+ and the establishment of a new equilibrium value, at about 65% of the unstimulated value. This corresponds to a decrease in K+ potential of about 15 mV. The effect of norepinephrine was stereospecific and reversible, and had an EC50 value of about 10 nM. As catecholamine effects were much more sensitive to phentolamine than to propranolol, the adrenergically-induced efflux was classified as predominantly alpha-adrenergic. It is suggested that the norepinephrine-induced K+ efflux is due to a (probably Ca2+-mediated) opening of K+ channels in the cell membrane, and that this effect occurs secondarily to the alpha-adrenergically induced membrane depolarization (and increase in cytosolic Ca2+). The increased PK over the cell membrane would counteract further depolarization, and the K+ gradient would then approach the Nernst equilibrium under the new steady-state conditions.
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| 16. |
- Nånberg, Eewa, 1957-, et al.
(författare)
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Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance of alpha-1-adrenergic stimulation
- 1985
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 844:1, s. 42-49
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Tidskriftsartikel (refereegranskat)abstract
- We have previously demonstrated mobilization of Ca2+ in and efflux of Rb+ (K+) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of the alpha 1-subtype. Further, we have found that the Rb+ (K+) efflux was dependent upon a primary Ca2+ mobilization event in response to the alpha 1-adrenergic stimulation, since the Rb+ efflux could also be demonstrated by the addition of the Ca2+ ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb+ effluxes were both inhibited by the Ca2+-dependent K+-channel blocker apamin. Apamin also significantly attenuated Ca2+ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that alpha 1-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca2+ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca2+ concentration which, in turn, activates a Ca2+-dependent K+ channel, leading to a K+ release from these cells. A possible role for this channel to sustain and augment the response to alpha 1-adrenergic stimulation is discussed.
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| 17. |
- Serrander, Lena, et al.
(författare)
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Particles binding β2-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis
- 1999
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 1452:2, s. 133-144
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Tidskriftsartikel (refereegranskat)abstract
- Complement-opsonised particles are readily ingested by human neutrophils through a complement receptor-mediated process leading to phagolysosome fusion and production of oxidative metabolites. To investigate the complement receptor 3 (CR3)-associated signal system involved, cells were challenged with protein A-positive, heat-killed Staphylococcus aureus to which antibodies with specificity for the subunits of the β2-integrins, i.e. anti-CD11b (the α subunit of CR3) and anti-CD18 (the β subunit of CR3), were bound through their Fc moiety. Despite not being ingested by the neutrophils, the surface associated anti-CD18- and anti-CD11b-coated particles were able to activate the neutrophil NADPH-oxidase. Also anti-CD11a- (the α subunit of LFA-1) and to a lesser extent anti-CD11c- (the α subunit of CR4) coated particles were able to trigger the NADPH-oxidase. The NADPH-oxidase was activated without extracellular release of reactive oxygen species. The activity was inhibited by cytochalasin B, suggesting a necessary role for the cytoskeleton in the signalling pathway that activates the oxidase. We show that particle-mediated cross-linking of β2-integrins on the neutrophil surface initiates a signalling cascade, involving cytoskeletal rearrangements, leading to an activation of the NADPH-oxidase without phagosome formation or extracellular release of reactive oxygen species.
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| 18. |
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| 19. |
- Al-Furoukh, Natalie, et al.
(författare)
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Binding to G-quadruplex RNA activates the mitochondrial GTPase NOA1
- 2013
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Ingår i: Biochimica Et Biophysica Acta-Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1833:12
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Tidskriftsartikel (refereegranskat)abstract
- NOA1 is an evolutionary conserved, nuclear encoded GTPase essential for mitochondrial function and cellular survival. The function of NOA1 for assembly of mitochondrial ribosomes and regulation of OXPHOS activity depends on its GTPase activity, but so far no ligands have been identified that regulate the GTPase activity of NOA1. To identify nucleic acids that bind to the RNA-binding domain of NOA1 we employed SELEX (Systemic Evolution of Ligands by Exponential Enrichment) using recombinant mouse wildtype NOA1 and the GTPase mutant NOA1-K353R We found that NOA1 binds specifically to oligonucleotides that fold into guanine tetrads (G-quadruplexes). Binding of G-quadruplex oligonucleotides stimulated the GTPase activity of NOA1 suggesting a regulatory link between G-quadruplex containing RNAs, NOA1 function and assembly of mitochondrial ribosomes. (C) 2013 Elsevier B.V. All rights reserved.
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| 20. |
- Al-Furoukh, Natalie, et al.
(författare)
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ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells
- 2015
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1853:10, s. 2580-2591
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Tidskriftsartikel (refereegranskat)abstract
- Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics. ClpX overexpression results in the upregulation of markers of the mitochondria( proteostasis pathway, known as the "mitochondrial unfolded protein response" (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153). In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.
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| 21. |
- Alajbegovic, Azra, et al.
(författare)
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Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization
- 2017
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Ingår i: Biochimica et Biophysica Acta - Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1864:6, s. 1088-1098
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Tidskriftsartikel (refereegranskat)abstract
- The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA.In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve.By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs.In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying vascular disease states, such as aortic aneurysm, as well as novel ideas regarding therapeutic strategies. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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| 22. |
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| 23. |
- Aspenström, Pontus
(författare)
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Integration of signalling pathways regulated by small GTPases and calcium
- 2004
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1742:1-3, s. 51-58
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Forskningsöversikt (refereegranskat)abstract
- The Ras superfamily of small GTPases constitutes a large group of structurally and functionally related proteins. They function as signalling switches in numerous signalling cascades in the cell. During the recent years, an increased awareness of a communication between signalling systems employing Ras-like GTPases and signalling systems employing calcium has emerged. For instance, the intensity of the activation of Ras-like GTPases is regulated by calcium-dependent mechanisms, acting on proteins that facilitate the activation or inactivation of the small GTPases. Other Ras-like GTPases have a direct influence on calcium signalling by regulating the activity of certain calcium channels. In addition, several small GTPases collaborate with calcium signalling in regulating cellular processes, such as cell adhesion, cell migration and exocytosis.
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| 24. |
- Autelli, Riccardo, et al.
(författare)
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Divergent pathways for TNF and C₂-ceramide toxicity in HTC hematoma cells
- 2009
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 1793, s. 1182-1190
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Tidskriftsartikel (refereegranskat)abstract
- We previously showed that, in the rat hepatoma cell line HTC, TNF brings about a non-caspase-dependent, apoptosis-like process requiring NADPH oxidase activity, an iron-mediated pro-oxidant status, and a functional acidic vacuolar compartment. This process may thus involve mechanisms such as autophagy or relocation of lysosomal enzymes, perhaps secondary to the formation of ceramide by acidic sphingomyelinase. Here we investigated whether ceramide formation contributes to the apoptogenic process. HTC cells were found to be sensitive to exogenous ceramide and significantly protected against TNF by desipramine, an inhibitor of lysosomal acid sphingomyelinase. However, Bcl-2 transfection and Bcl-x(L) upregulation by dexamethasone significantly diminished the apoptogenic effect of ceramide but not that of TNF, suggesting that ceramide is not directly involved in TNF toxicity. Moreover, Bcl-x(L) silencing precluded dexamethasone-induced protection against ceramide and, by itself, induced massive death, demonstrating the strict dependence of HTC cells on Bcl-x(L) for survival also under standard culture conditions.
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| 25. |
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| 26. |
- Ballester-Tomás, Lidia, et al.
(författare)
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Inappropriate translation inhibition and P-body formation cause cold-sensitivity in tryptophan-auxotroph yeast mutants
- 2017
-
Ingår i: Biochimica et Biophysica Acta - Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1864, s. 314-323
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Tidskriftsartikel (refereegranskat)abstract
- © 2016 Elsevier B.V. In response to different adverse conditions, most eukaryotic organisms, including Saccharomyces cerevisiae, downregulate protein synthesis through the phosphorylation of eIF2α (eukaryotic initiation factor 2α) by Gcn2, a highly conserved protein kinase. Gcn2 also controls the translation of Gcn4, a transcription factor involved in the induction of amino acid biosynthesis enzymes. Here, we have studied the functional role of Gcn2 and Gcn2-regulating proteins, in controlling translation during temperature downshifts of TRP1 and trp1 yeast cells. Our results suggest that neither cold-instigated amino acid limitation nor Gcn2 are involved in the translation suppression at low temperature. However, loss of TRP1 causes increased eIF2α phosphorylation, Gcn2-dependent polysome disassembly and overactivity of Gcn4, which result in cold-sensitivity. Indeed, knock-out of GCN2 improves cold growth of trp1 cells. Likewise, mutation of several Gcn2-regulators and effectors results in cold-growth effects. Remarkably, we found that Hog1, the osmoresponsive MAPK, plays a role in the regulatory mechanism of Gcn2-eIF2α. Finally, we demonstrated that P-body formation responds to a downshift in temperature in a TRP1-dependent manner and is required for cold tolerance.
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| 27. |
- Barajas-Lopez, Juan de Dios, et al.
(författare)
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Plastid-to-nucleus communication, signals controlling the running of the plant cell
- 2013
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1833:2, s. 425-437
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Forskningsöversikt (refereegranskat)abstract
- The presence of genes encoding organellar proteins in both the nucleus and the organelle necessitates tight coordination of expression by the different genomes, and this has led to the evolution of sophisticated intracellular signaling networks. Organelle-to-nucleus signaling, or retrograde control, coordinates the expression of nuclear genes encoding organellar proteins with the metabolic and developmental state of the organelle. Complex networks of retrograde signals orchestrate major changes in nuclear gene expression and coordinate cellular activities and assist the cell during plant development and stress responses. It has become clear that, even though the chloroplast depends on the nucleus for its function, plastid signals play important roles in an array of different cellular processes vital to the plant. Hence, the chloroplast exerts significant control over the running of the cell. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.
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| 28. |
- Baxtera, Shannon A., et al.
(författare)
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Regulation of the lymphatic endothelial cell cycle by the PROX1 homeodomain protein
- 2011
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1813:1, s. 201-212
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Tidskriftsartikel (refereegranskat)abstract
- The homeobox transcription factor PROX1 is essential for the development and maintenance of lymphatic vasculature. How PROX1 regulates lymphatic endothelial cell fate remains undefined. PROX1 has been shown to upregulate the expression of Cyclin E, which mediates the G1 to S transition of the cell cycle. Here we demonstrate that PROX1 activates the mouse Cyclin E1 (Ccne1) promoter via two proximal E2F-binding sites. We have determined that the N-terminal region of PROX1 is sufficient to activate a 1-kb Ccne1 promoter, whereas the homeodomain is dispensable for activation. We have identified that the Prospero domain 1 (PD1) is required for the nuclear localization of PROX1. Our comparison of two DNA-binding-deficient constructs of PROX1 showed a cell-type-specific difference between these two proteins in both their localization and function. We demonstrated that siRNA-mediated knockdown of PROX1 in lymphatic endothelial cells decreases progression from G1 to S phase of the cell cycle. We conclude that PROX1 activates the Ccne1 promoter independent of DNA binding, and our results illustrate a novel role for PROX1 in the regulation of lymphatic endothelial cell proliferation.
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| 29. |
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| 30. |
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| 31. |
- Bodvard, Kristofer, 1981, et al.
(författare)
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Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p.
- 2011
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1813:2, s. 358-366
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Tidskriftsartikel (refereegranskat)abstract
- Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters. We here investigate how continuous illumination with blue light, corresponding to GFP excitation wavelengths, affects the localization pattern of Msn2p-GFP in budding yeast. The localization pattern was analyzed using a novel approach that combines wavelet decomposition and change point analysis. It was found that the Msn2p nucleocytoplasmic localization trajectories for individual cells exhibit up to three distinct and successive states; i) Msn2p localizes to the cytoplasm; ii) Msn2p rapidly shuttles between the cytoplasm and the nucleus; iii) Msn2p localizes to the nucleus. Many cells pass through all states consecutively at high light intensities, while at lower light intensities most cells only reach states i) or ii). This behaviour strongly indicates that continuous light exposure gradually increases the stress level over time, presumably through continuous accumulation of toxic photoproducts, thereby forcing the cell through a bistable region corresponding to nucleocytoplasmic oscillations. We also show that the localization patterns are dependent on protein kinase A (PKA) activity, i.e. yeast cells with constantly low PKA activity showed a stronger stress response. In particular, the nucleocytoplasmic oscillation frequency was found to be significantly higher for cells with low PKA activity for all light intensities.
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| 32. |
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| 33. |
- Boström, Elisabeth Almer, 1983, et al.
(författare)
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Resistin is stored in neutrophil granules being released upon challenge with inflammatory stimuli.
- 2009
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1793:12, s. 1894-900
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Tidskriftsartikel (refereegranskat)abstract
- We have recently shown that resistin is a key mediator of arthritis accumulating in the inflamed joints and exerting its pro-inflammatory properties independently of TNFalpha. Here we evaluate neutrophils as a cellular source of resistin. Human neutrophils were subjected to subcellular fractionation where the presence of resistin was assessed using western blot, ELISA, and mass spectrometry. Presence of resistin on the neutrophil surface was visualized by flow cytometry. More than 95% of the neutrophils in circulation and in synovial fluid express resistin on their surface. Stimulation of mature neutrophils with fMLF induced release of resistin into supernatants and increased expression of resistin on the surface. Resistin is mobilized simultaneously with lactoferrin, a protein found in specific granules, and with granule-stored CR3/CD11b. Subcellular fractionation of human neutrophils demonstrated the presence of resistin in azurophilic and in specific granules. Here we show that neutrophils have two pools of resistin, the major one exists in specific granules, and the second on their cell membrane. Release of resistin from the neutrophil granules probably serves the main source of resistin at the site of inflammation.
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| 34. |
- Chen, D., et al.
(författare)
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GSTpi protects against angiotensin II-induced proliferation and migration of vascular smooth muscle cells by preventing signal transducer and activator of transcription 3 activation
- 2014
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1843:2, s. 454-463
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Tidskriftsartikel (refereegranskat)abstract
- Angiotensin II (Ang II)-elicited excessive proliferation, hypertrophy and migration of vascular smooth muscle cells (VSMCs) are vital to the pathogenesis of atheroclerosis. Glutathione S-transferase pi (GSTpi) exists extensively in various kinds of cells and protects cells against different stresses. However, knowledge remains limited about what GSTpi acts in VSMCs. We investigated the effect of GSTpi on Ang II-induced VSMC proliferation, hypertrophy and migration and its latent mechanism. Overexpression and RNAi experiments demonstrated that GSTpi inhibited Ang II-induced proliferation, hypertrophy and migration of VSMCs and arrested progression of cell cycle from G0/G1 to S phase. Immunoprecipitation, mass spectrometry and confocal microscopy analyses showed that GSTpi directly associated with signal transducer and activator of transcription 3 (STAT3) to prevent Ang II-triggered binding of Src to STAT3 and thus suppressed Ang II-stimulated phosphorylation and nuclear translocation of STAT3, as well as cyclin D1 expression. In contrast, GSTpi didn't affect Ang II-activated extracellular signal-regulated kinase (ERK1/2). GSTpi acts as a negative regulator to prevent Ang II-triggered proliferative signaling in VSMCs, suggesting that it may protect vessels against the stresses associated with atherosclerosis formation.
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| 35. |
- Ciesla, Malgorzata, et al.
(författare)
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Fructose bisphosphate aldolase is involved in the control of RNA polymerase III-directed transcription
- 2014
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1843:6, s. 1103-1110
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Tidskriftsartikel (refereegranskat)abstract
- Yeast Fba1 (fructose 1,6-bisphosphate aldolase) is a glycolytic enzyme essential for viability. The overproduction of Fba1 enables overcoming of a severe growth defect caused by a missense mutation rpc128-1007 in a gene encoding the 028 protein, the second largest subunit of the RNA polymerase III complex. The suppression of the growth phenotype by Fbal is accompanied by enhanced de novo tRNA transcription in rpc128-1007 cells. We inactivated residues critical for the catalytic activity of Fbal. Overproduction of inactive aldolase still suppressed the rpc128-1007 phenotype, indicating that the function of this glycolytic enzyme in RNA polymerase III transcription is independent of its catalytic activity. Yeast Fbal was determined to interact with the RNA polymerase III complex by coimmunoprecipitation. Additionally, a role of aldolase in control of tRNA transcription was confirmed by ChIP experiments. The results indicate a novel direct relationship between RNA polymerase HI transcription and aldolase.
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| 36. |
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| 37. |
- Damdimopoulos, Anastasios E., et al.
(författare)
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Nuclear immobilization of DsRed1 tagged proteins : a novel tool for studying DNA-protein interactions?
- 2007
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 1773:6, s. 687-690
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Tidskriftsartikel (refereegranskat)abstract
- DsRed1 is a red fluorescent protein that can be used as a fusion partner with other proteins to determine their subcellular localization, similarly to the popular green fluorescent proteins (GFP). Here, we report that fusion of DsRed1 to estrogen receptor alpha (ER alpha) renders the transcription factor immobile within the nucleus. Furthermore, we show that the immobilization is dependent on DNA interaction and that the binding to the DNA can be direct as well as indirect for DsRed to immobilize with its fusion partners. This observation could provide a new tool to be used for the identification of target genes containing low affinity binding sites for several transcription factors including ER alpha. In addition, it could be employed for studies on protein-DNA interactions as well as protein-protein interactions during protein complex formation on chromatin in the event of transcription initiation and regulation.
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| 38. |
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| 39. |
- Daniel, Geoffrey
(författare)
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γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks
- 2018
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Ingår i: Biochimica et biophysica acta. Molecular cell research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1865, s. 734-748
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Tidskriftsartikel (refereegranskat)abstract
- gamma-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of gamma-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of gamma-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular gamma-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified gamma-tubulin free of gamma-tubulin complex proteins (GCPs) was demonstrated for both plant and human gamma-tubulin. Moreover, gamma-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of gamma-tubulin to oligomerize/polymerize was supported by conservation of alpha- and beta-tubulin interfaces for longitudinal and lateral interactions for gamma-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short gamma-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of gamma-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed gamma-tubulin location in acentrosomal plant cells as well as at sites of local gamma-tubulin enrichment after drug treatment. Our findings that gamma-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, gamma-tubulin may also have scaffolding or sequestration functions.
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| 40. |
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| 41. |
- Elo, Mika, et al.
(författare)
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Hsp90 inhibitor geldanamycin increases hsp70 mRNA stabilisation but fails to activate HSF1 in cells exposed to hydrostatic pressure.
- 2005
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434. ; 1743:1-2, s. 115-119
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Tidskriftsartikel (refereegranskat)abstract
- High hydrostatic pressure (HP) increases Hsp70 protein and mRNA levels by increasing the mRNA half-life without activation of HSF1 transcription factor. We investigated whether this change in gene expression requires Hsp90, previously shown to regulate hsp70 genes via HSF1. In HeLa cells, both HP and Hsp90 inhibitor geldanamycin (GA) up-regulated Hsp70 expression through mRNA stabilisation. GA, unlike HP, increased HSF1 activation. However, when exposures were used together a marked Hsp70 response was observed with mRNA stabilisation without coincidence of HSF1 activation. Our data suggests that Hsp90 is involved in hsp70 mRNA stabilisation and the HSF1 activation can be suppressed by high HP.
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| 42. |
- Erlandsson, Malin, 1972, et al.
(författare)
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Expression of metastasin S100A4 is essential for bone resorption and regulates osteoclast function.
- 2013
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1833:12, s. 2653-2663
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Tidskriftsartikel (refereegranskat)abstract
- S100A4 is a Ca-binding protein that regulates cell growth, survival, and motility. The abundant expression of S100A4 in rheumatiod arthritis contributes to the invasive growth of joint tissue and to bone damage. In the present study, we analysed the role of S100A4 in bone homeostasis.
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| 43. |
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| 44. |
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| 45. |
- Forsman, Huamei, et al.
(författare)
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Structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a formylmethionyl group.
- 2015
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1853:1, s. 192-200
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Tidskriftsartikel (refereegranskat)abstract
- Pathogenic Staphylococcus aureus strains produce N-formylmethionyl containing peptides, of which the tetrapeptide fMIFL is a potent activator of the neutrophil formyl peptide receptor 1 (FPR1) and the PSMα2 peptide is a potent activator of the closely related FPR2. Variants derived from these two peptide activators were used to disclose the structural determinants for receptor interaction. Removal of five amino acids from the C-terminus of PSMα2 gave rise to a peptide that had lost the receptor-independent neutrophil permeabilizing effect, whereas neutrophil activation capacity as well as its preference for FPR2 was retained. Shorter peptides, PSMα21-10 and PSMα21-5, activate neutrophils, but the receptor preference for these peptides was switched to FPR1. The fMIFL-PSM5-16 peptide, in which the N-terminus of PSMα21-16 was replaced by the sequence fMIFL, was a dual agonist for FPR1/FPR2, whereas fMIFL-PSM5-10 preferred FPR1 to FPR2. Further, an Ile residue was identified as a key determinant for interaction with FPR2. A chimeric receptor in which the cytoplasmic tail of FPR1 was replaced by the corresponding part of FPR2 lost the ability to recognize FPR1 agonists, but gained function in relation to FPR2 agonists. Taken together, our data demonstrate that the C-terminus of the PSMα2 peptide plays a critical role for its cytotoxicity, but is not essential for the receptor-mediated pro-inflammatory activity. More importantly, we show that the amino acids present in the C-terminus, which are not supposed to occupy the agonist-binding pocket in the FPRs, are of importance for the choice of receptor.
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| 46. |
- Forsman, Huamei, et al.
(författare)
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The leukocyte chemotactic receptor FPR2, but not the closely related FPR1, is sensitive to cell-penetrating pepducins with amino acid sequences descending from the third intracellular receptor loop.
- 2013
-
Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1833:8, s. 1914-1923
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Tidskriftsartikel (refereegranskat)abstract
- Lipidated peptides (pepducins) can activate certain G-protein coupled receptors (GPCRs) through a unique allosteric modulation mechanism involving cytosolic receptor domains. Pepducins with the amino acid sequence of the third intracellular loop of the neutrophil formyl peptide receptors (FPRs) as a common denominator were N-terminally conjugated with palmitic acid. F2Pal16, containing the 16 amino acids present in the third intracellular loop of FPR2, induced superoxide production in human neutrophils and the activity was sensitive to FPR2 antagonists. Cells over-expressing FPR2 were similarly responsive and responded with a transient increase in cytosolic calcium. No such effects were observed with the corresponding FPR1 pepducin. The peptide alone, lacking palmitic acid, did not activate neutrophils. A ten amino acid long pepducin F2Pal10, that was a more potent neutrophil activator than F2Pal16, was used for amino acid substitution studies. The sequences of FPR1 and FPR2 in the third intracellular loop differ by only two amino acids, and a pepducin with the FPR2-specific K231 replaced by the FPR1-specific Q231 lost all activity. The active F2Pal10 pepducin also triggered a response in cells expressing a mutated FPR2 with the third intracellular loop identical to that of FPR1. The data presented suggest that the same signaling pathways are activated when the signaling cascade is initiated by a classical receptor agonist (outside-in signaling) and when signaling starts on the cytosolic side of the membrane by a pepducin (inside-in signaling). A fundamental difference is also disclosed between the two neutrophil FPRs regarding their sensitivities to third intracellular loop pepducins.
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| 47. |
- Fredriksson, Johanna, et al.
(författare)
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GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by beta-arrestin recruiting GPR84 agonists
- 2022
-
Ingår i: Biochimica Et Biophysica Acta-Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1869:7
-
Tidskriftsartikel (refereegranskat)abstract
- In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84-but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by beta-arrestin recruitment data. The ROS production induced by a non beta-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this beta-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with beta-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils.
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| 48. |
- Gabl, Michael, et al.
(författare)
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A pepducin designed to modulate P2Y2R function interacts with FPR2 in human neutrophils and transfers ATP to an NADPH-oxidase-activating ligand through a receptor cross-talk mechanism.
- 2016
-
Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1863:6 Pt A, s. 1228-37
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Tidskriftsartikel (refereegranskat)abstract
- Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y2R agonist ATP, the P2Y2PalIC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y2R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y2R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y2R (P2Y2PalIC3) exhibited the same basic functions as P2Y2PalIC2, whereas one with a peptide that was identical to the first intracellular loop (P2Y2PalIC1) lacked these functions. The responses induced in neutrophils by the P2Y2R pepducins were not inhibited by the P2Y2R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y2R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y2R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y2R activate human neutrophils through FPR2. We also show that the P2Y2PalIC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y2R/ATP and FPR2/P2Y2-pepducin.
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| 49. |
- Ghavami, Saeid, et al.
(författare)
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Airway mesenchymal cell death by mevalonate cascade inhibition : integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins
- 2014
-
Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1843:7, s. 1259-1271
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Tidskriftsartikel (refereegranskat)abstract
- HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1 alpha) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX(-/-)/BAIC(-/) murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.
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| 50. |
- Ghavami, Saeid, et al.
(författare)
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Autophagy regulates trans fatty acid-mediated apoptosis in primary cardiac myofibroblasts.
- 2012
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier BV. - 0167-4889 .- 1879-2596. ; 1823:12, s. 2274-2286
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Tidskriftsartikel (refereegranskat)abstract
- Trans fats are not a homogeneous group of molecules and less is known about the cellular effects of individual members of the group. Vaccenic acid (VA) and elaidic acid (EA) are the predominant trans monoenes in ruminant fats and vegetable oil, respectively. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofibroblasts (rVF). The MTT assay demonstrated that both VA and EA (200μM, 0-72h) reduced cell viability in rVF (P<0.001). The FACS assay confirmed that both VA and EA induced apoptosis in rVF, and this was concomitant with elevation in cleaved caspase-9, -3 and -7, but not caspase-8. VA and EA decreased the expression ratio of Bcl2:Bax, induced Bax translocation to mitochondria and decrease in mitochondrial membrane potential (Δψ). BAX and BAX/BAK silencing in mouse embryonic fibroblasts (MEF) inhibited VA and EA-induced cell death compared to the corresponding wild type cells. Transmission electron microscopy revealed that VA and EA also induced macroautophagosome formation in rVF, and immunoblot analysis confirmed the induction of several autophagy markers: LC3-β lipidation, Atg5-12 accumulation, and increased beclin-1. Finally, deletion of autophagy genes, ATG3 and ATG5 significantly inhibited VA and EA-induced cell death (P<0.001). Our findings show for the first time that trans fat acid (TFA) induces simultaneous apoptosis and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this pro-apoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.
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