| 1. |
- Baird, Sarah K, et al.
(författare)
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Metallothionein protects against oxidative stress-induced lysosomal destabilization
- 2006
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 394:1, s. 275-283
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Tidskriftsartikel (refereegranskat)abstract
- The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions. © 2006 Biochemical Society.
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| 2. |
- Berndt, Carsten, et al.
(författare)
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Ascorbate and endocytosed Motexafin gadolinium induce lysosomal rupture.
- 2011
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Ingår i: Cancer Letters. - : Elsevier BV. - 0304-3835 .- 1872-7980. ; 307:2, s. 119-23
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Tidskriftsartikel (refereegranskat)abstract
- Motexafin gadolinium (MGd) sensitizes malignant cells to ionizing radiation, although the underlying mechanisms for uptake and sensitization are both unclear. Here we show that MGd is endocytosed by the clathrin-dependent pathway with ensuing lysosomal membrane permeabilization, most likely via formation of reactive oxygen species involving redox-active metabolites, such as ascorbate. We propose that subsequent apoptosis is a synergistic effect of irradiation and high MGd concentrations in malignant cells due to their pronounced endocytic activity. The results provide novel insights into the mode of action of this promising anti-cancer drug, which is currently under clinical trials.
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| 3. |
- Berndt, Carsten, et al.
(författare)
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Chelation of lysosomal iron protects against ionizing radiation.
- 2010
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 432:2, s. 295-301
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Tidskriftsartikel (refereegranskat)abstract
- Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.
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| 4. |
- Dong, X.-P., et al.
(författare)
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The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel
- 2008
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 455:7215, s. 992-996
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Tidskriftsartikel (refereegranskat)abstract
- TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe3+-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe2+ transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe2+ transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe2+ permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe2+ at varying degrees, which correlate well with the disease severity. A comparison of TRPML1-/-ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe2+ levels, an increase in intralysosomal Fe 2+ levels and an accumulation of lipofuscin-like molecules in TRPML1-/- cells. We propose that TRPML1 mediates a mechanism by which Fe2+ is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients. ©2008 Macmillan Publishers Limited. All rights reserved.
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| 5. |
- Karlsson, Jan Olof G, et al.
(författare)
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Superior Therapeutic Index of Calmangafodipir in Comparison to Mangafodipir as a Chemotherapy Adjunct
- 2012
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Ingår i: TRANSLATIONAL ONCOLOGY. - : NEOPLASIA PRESS, 1150 W MEDICAL CENTER DR, MSRB III, RM 9303, ANN ARBOR, MI 48109-0648 USA. - 1944-7124 .- 1936-5233. ; 5:6, s. 492-502
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Tidskriftsartikel (refereegranskat)abstract
- Mangafodipir is a magnetic resonance imaging contrast agent with manganese superoxide dismutase (MnSOD) mimetic activity. The MnSOD mimetic activity protects healthy cells against oxidative stress-induced detrimental effects, e. g., myelosuppressive effects of chemotherapy drugs. The contrast property depends on in vivo dissociation of Mn2+ from mangafodipir-about 80% dissociates after injection. The SOD mimetic activity, however, depends on the intact Mn complex. Complexed Mn2+ is readily excreted in the urine, whereas dissociated Mn2+ is excreted slowly via the biliary route. Mn is an essential but also a potentially neurotoxic metal. For more frequent therapeutic use, neurotoxicity due to Mn accumulation in the brain may represent a serious problem. Replacement of 4/5 of Mn2+ in mangafodipir with Ca2+ (resulting in calmangafodipir) stabilizes it from releasing Mn2+ after administration, which roughly doubles renal excretion of Mn. A considerable part of Mn2+ release from mangafodipir is governed by the presence of a limited amount of plasma zinc (Zn2+). Zn2+ has roughly 10(3) and 10(9) times higher affinity than Mn2+ and Ca2+, respectively, for fodipir. Replacement of 80% of Mn2+ with Ca2+ is enough for binding a considerable amount of the readily available plasma Zn2+, resulting in considerably less Mn2+ release and retention in the brain and other organs. At equivalent Mn2+ doses, calmangafodipir was significantly more efficacious than mangafodipir to protect BALB/c mice against myelosuppressive effects of the chemotherapy drug oxaliplatin. Calmangafodipir did not interfere negatively with the antitumor activity of oxaliplatin in CT26 tumor-bearing syngenic BALB/c mice, contrary calmangafodipir increased the antitumor activity. Translational Oncology (2012) 5, 492-502
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| 6. |
- Karlsson, Markus, et al.
(författare)
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Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress
- 2016
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Ingår i: Acta Ophthalmologica. - : Wiley-Blackwell Publishing Inc.. - 1755-375X .- 1755-3768. ; 94:6, s. 556-565
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Tidskriftsartikel (refereegranskat)abstract
- PurposeOxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress.MethodsThe sensitivity of ARPE-19 cells to H2O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4, heat shock or FeCl3, as well as siRNA-mediated downregulation of the same proteins.ResultsUpregulation of MT, HSP70 and FT did not improve survival following exposure to H2O2. This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins.ConclusionThe findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low.
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| 7. |
- Karlsson, Markus, et al.
(författare)
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Autophagy of iron-binding proteins may contribute to the oxidative stress resistance of ARPE-19 cells
- 2013
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Ingår i: Experimental Eye Research. - : Elsevier. - 0014-4835 .- 1096-0007. ; 116, s. 359-365
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to elucidate possible reasons for the remarkable resistance of human retinal pigment epithelial (RPE) cells to oxidative stress. Much oxidative damage is due to hydrogen peroxide meeting redox-active iron in the acidic and reducing lysosomal environment, resulting in the production of toxic hydroxyl radicals that may oxidize intralysosomal content, leading to lipofuscin (LF) formation or, if more extensive, to permeabilization of lysosomal membranes. Formation of LF is a risk factor for age-related macular degeneration (AMD) and known to jeopardize normal autophagic rejuvenation of vital cellular biomolecules. Lysosomal membrane permeabilization causes release of lysosomal content (redox-active iron, lytic enzymes), which may then cause cell death. Total cellular and lysosomal low-mass iron of cultured, immortalized human RPE (ARPE-19) cells was compared to that of another professional scavenger cell line, J774, using atomic absorption spectroscopy and the cytochemical sulfide-silver method (SSM). It was found that both cell lines contained comparable levels of total as well as intralysosomal iron, suggesting that the latter is mainly kept in a non-redox-active state in ARPE-19 cells. Basal levels and capacity for upregulation of the iron-binding proteins ferritin, metallothionein and heat shock protein 70 were tested in both cell lines using immunoblotting. Compared to J774 cells, ARPE-19 cells were found to contain very high basal levels of all these proteins, which could be even further upregulated following appropriate stimulation. These findings suggest that a high basal expression of iron-binding stress proteins, which during their normal autophagic turnover in lysosomes may temporarily bind iron prior to their degradation, could contribute to the unusual oxidative stress-resistance of ARPE-19 cells. A high steady state influx of such proteins into lysosomes would keep the level of lysosomal redox-active iron permanently low. This, in turn, should delay intralysosomal accumulation of LF in RPE cells, which is known to reduce autophagic turnover as well as uptake and degradation of worn out photoreceptor tips. This may explain why severe LF accumulation and AMD normally do not develop until fairly late in life, in spite of RPE cells being continuously exposed to high levels of oxygen and light, as well as large amounts of lipid-rich material.
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| 8. |
- Karlsson, Markus
(författare)
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Oxidative stress-related damage of retinal pigment epithelial cells : possible protective properties of autophagocytosed iron-binding proteins
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxidative stress is a major pathogenic factor in the development of age-related macular degeneration (AMD), which is the most common cause of severe central visual impairment in the elderly population in the western world.It is believed that the degenerative process starts in the retinal pigment epithelium (RPE). The post-mitotic RPE is a single layer of pigmented cells located behind the photoreceptors – rods and cones – of the retina. Daily, the RPE cells phagocytose and recycle the expended tips of the photoreceptor outer segments. This heavy phagocytic burden leads to substantial oxidative stress in the cells, which is further enhanced by intense illumination and a high oxygen tension. A hallmark of early AMD is a progressive build-up of the non-degradable age pigment lipofuscin (LF) in lysosomes of the RPE. LF accumulation hampers phagocytosis and autophagy in the RPE, resulting in increased amounts of cellular debris in and around the cells. This decreases the function and viability of both RPE cells and photoreceptors.Iron is known to accumulate in the retina with increasing age, particularly in AMDaffected eyes, and amplifies oxidative stress by acting as a potent catalyst in the generation of hydroxyl radicals. These highly reactive radicals contribute to LF formation and may, if abundantly present, also directly damage lysosomal membranes. The subsequent leakage of degrading enzymes to the cytosol initiates cell death via apoptosis or necrosis.In this thesis, we have investigated the oxidative stress response of human RPE (ARPE-19) cells compared to murine J774 cells, another type of lysosome-rich cells with a high phagocytic capacity. The ARPE-19 cells were found to be extremely resistant to oxidative stress and tolerated exposure to single doses of H2O2 in concentrations up to 150 times higher than the J774 cells before lysosomal rupture and ensuing cell death occurred. This resistance was increased even further when the cells were protected with a potent iron chelator that prevents redox-active iron to participate in hydroxyl radical generation. Both cell lines were shown to be equally effective in degrading H2O2 and seem to contain comparable amounts of total as well as intralysosomal iron. Therefore, we reasoned that the insensitivity of ARPE-19 cells to H2O2 exposure might be related to a mechanism which keeps their intralysosomal iron bound in a non redox-active form. This theory was supported by our finding of very high basal expression levels of metallothionein (MT), heat shock-protein 70 (HSP70) and ferritin (FT) in ARPE-19 cells compared to J774 cells. All of these proteins have previously been shown to possess potent iron-binding properties. The ARPE-19 cells were also shown to have a higher basal rate of autophagy. SiRNA-mediated attenuation of MT, HSP70 and FT levels in the ARPE-19 cells resulted, to some degree, in an increased sensitivity to H2O2 treatment. Furthermore, a human cell stress array showed several other stress-related proteins to be up-regulated in ARPE-19 cells.Additionally, we evaluated the commonly used, but frequently misinterpreted, H2DCF test for oxidative stress. It was demonstrated that oxidation of H2DCF into fluorescent DCF mainly reflects relocation to the cytosol of lysosomal iron and mitochondrial cytochrome c, rather than being the result of some poorly defined “general” oxidative stress.In conclusion, our results indicate that the extreme resistance to oxidative stress exhibited by the ARPE-19 cells might be related to a high continuous autophagic influx of iron-binding proteins into the lysosomal compartment. Before being degraded, such proteins will temporarily keep intralysosomal iron bound in a non redox-active form, thereby inhibiting hydroxyl radical formation. This may partly explain why RPE cells, in spite of their exposed location and heavy burden of phagocytosis, usually manage to survive and evade significant LF accumulation until late in life.
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| 9. |
- Karlsson, Markus, et al.
(författare)
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What does the commonly used DCF test for oxidative stress really show?
- 2010
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 428:2, s. 183-90
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Tidskriftsartikel (refereegranskat)abstract
- H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.
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| 10. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 11. |
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| 12. |
- Kurz, Tino, et al.
(författare)
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A myelopoiesis gene signature during remission in ANCA associated vasculitis does not predict relapses but seems to reflect ongoing prednisolone therapy
- 2014
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Ingår i: Clinical and Experimental Immunology. - : Wiley-Blackwell. - 0009-9104 .- 1365-2249. ; 175:2, s. 215-226
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Tidskriftsartikel (refereegranskat)abstract
- A myelopoiesis gene signature in circulating leucocytes, exemplified by increased myeloperoxidase (MPO) and proteinase 3 (PR3) mRNA levels, has been reported in patients with active anti-neutrophil cytoplasm antibody associated vasculitis (AAV) and to a lesser extent during remission. We hypothesized that this signature could predict disease relapse. mRNA levels of PR3, MPO, selected myelopoiesis transcription factors (CEBPA, CEBPB, SPIB, SPI1) and microRNAs (miRNAs) from patient and control peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) were analyzed and associated with clinical data. Patients in stable remission had higher mRNA levels for PR3 (PBMCs, PMNs) and MPO (PBMCs). PR3 and SPIB mRNA correlated positively in control but negatively in patient PBMCs. Statistically significant correlations existed between PR3 mRNA and several miRNAs in controls, but not in patients. PR3/MPO mRNA levels were not associated with previous or future relapses but correlated to steroid treatment. Prednisolone doses were negatively linked to SPIB and miR-155-5p, miR-339-5p (PBMCs) and to miR-221, miR-361, miR-505 (PMNs). PR3 mRNA in PBMCs correlated with time since last flare, blood leucocyte count and estimated glomerular filtration rate. Our results show that elevated leucocyte PR3 mRNA levels in AAV patients in remission do not predict relapse. The origin seems multifactorial, but to an important part explainable by prednisolone action. Gene signatures in patients with AAV undergoing steroid treatment should therefore be interpreted accordingly.
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| 13. |
- Kurz, Tino, et al.
(författare)
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ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron
- 2009
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Ingår i: AUTOPHAGY. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 5:4, s. 494-501
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Tidskriftsartikel (refereegranskat)abstract
- Normal retinal pigment epithelial (RPE) cells are postmitotic, long-lived and basically not replaced. Daily, they phagocytose substantial amounts of lipid-rich material (photoreceptor outer segment discs), and they do so in the most oxygenated part of the body-the retina. One would imagine that this state of affairs should be associated with a rapid formation of the age pigment lipofuscin (LF). However, LF accumulation is slow and reaches significant amounts only late in life when, if substantial, it often coincides with or causes age-related macular degeneration. LF formation occurs inside the lysosomal compartment as a result of iron-catalyzed peroxidation and polymerization. This process requires phagocytosed or autophagocytosed material under degradation, but also the presence of redox-active low mass iron and hydrogen peroxide. To gain some information on how RPE cells are able to evade LF formation, we investigated the response of immortalized human RPE cells (ARPE-19) to oxidative stress with/without the protection of a strong iron-chelator. The cells were found to be extremely resistant to hydrogen peroxide-induced lysosomal rupture and ensuing cell death. This marked resistance to oxidative stress was not explained by enhanced degradation of hydrogen peroxide, but to a certain extent further increased by the potent lipophilic iron chelator STH. The cells were also able to survive, and even replicate, at high concentrations of SIH and showed a high degree of basal autophagic flux. We hypothesize that RPE cells have a highly developed capacity to keep lysosomal iron in a nonredox-active form, perhaps by pronounced autophagy of iron-binding proteins in combination with an ability to rapidly relocate low mass iron from the lysosomal compartment.
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| 14. |
- Kurz, Tino, 1974-, et al.
(författare)
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Autophagy, ageing and apoptosis : The role of oxidative stress and lysosomal iron
- 2007
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 462:2, s. 220-230
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Tidskriftsartikel (refereegranskat)abstract
- As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells-probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term. © 2007 Elsevier Inc. All rights reserved.
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| 15. |
- Kurz, Tino, et al.
(författare)
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Autophagy of HSP70 and chelation of lysosomal iron in a non-redox-active form
- 2009
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Ingår i: AUTOPHAGY. - 1554-8627. ; 5:1, s. 93-95
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Tidskriftsartikel (refereegranskat)abstract
- Lysosomes contain most of the cells supply of labile iron, which makes them sensitive to oxidative stress. To keep lysosomal labile iron at a minimum, a cellular strategy might be to autophagocytose iron-binding proteins that temporarily would chelate iron in a nonredox-active form. Previously we have shown that autophagy of metallothioneins, as well as of non-Fe-saturated ferritin, meets this goal. Here we add another stress-regulated protein to the list, namely HSP70.
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| 16. |
- Kurz, Tino
(författare)
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Can lipofuscin accumulation be prevented?
- 2008
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Ingår i: Rejuvenation Research. - : Mary Ann Liebert Inc. - 1549-1684 .- 1557-8577. ; 11:2, s. 441-443
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Tidskriftsartikel (refereegranskat)abstract
- During normal autophagic degradation of mitochondria and iron-containing proteins in lysosomes, iron is released intralysosomally where it may react with hydrogen peroxide forming hydroxyl radicals (Fenton reaction). Depending on their rate of formation, these highly reactive radicals can cross-link intralysosomal material, leading to lipofuscin formation, or destabilize the lysosomal membrane, which induces apoptosis/necrosis. Since the sensitivity of lysosomes to oxidative stress can be manipulated by altering the intralysosomal level of redox-active iron, it follows that lipofuscin formation might also be influenced. It is suggested that pulse doses of iron chelators that easily penetrate membranes could be used to diminish lipofuscinogenesis. © 2008 Mary Ann Liebert, Inc.
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| 17. |
- Kurz, Tino, et al.
(författare)
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Cell sensitivity to oxidative stress is influenced by ferritin autophagy
- 2011
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Ingår i: FREE RADICAL BIOLOGY AND MEDICINE. - : Elsevier Science B.V., Amsterdam.. - 0891-5849. ; 50:11, s. 1647-1658
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Tidskriftsartikel (refereegranskat)abstract
- To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3 h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.
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| 18. |
- Kurz, Tino, et al.
(författare)
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Effects of MnDPDP andICRF-187 on Doxorubicin-Induced Cardiotoxicityand Anticancer Activity1
- 2012
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Ingår i: Translational Oncology. - : Elsevier BV. - 1944-7124 .- 1936-5233. ; 5:4, s. 252-259
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress participates in doxorubicin (Dx)–induced cardiotoxicity. The metal complex MnDPDP and its metaboliteMnPLED possess SOD-mimetic activity, DPDP and PLED have, in addition, high affinity for iron. Mice wereinjected intravenously with MnDPDP, DPDP, or dexrazoxane (ICRF-187). Thirty minutes later, mice were killed, theleft atria were hung in organ baths and electrically stimulated, saline or Dx was added, and the contractility wasmeasured for 60 minutes. In parallel experiments, 10 μM MnDPDP or MnPLED was added directly into the organbath. The effect of MnDPDP on antitumor activity of Dx against two human tumor xenografts (MX-1 and A2780)was investigated. The in vitro cytotoxic activity was studied by co-incubating A2780 cells with MnDPDP, DPDP,and/or Dx. Dx caused a marked reduction in contractile force. In vivo treatment with MnDPDP and ICRF-187 attenuatedthe negative effect of Dx. When added directly into the bath, MnDPDP did not protect, whereas MnPLEDattenuated the Dx effect by approximately 50%. MnDPDP or ICRF-187 did not interfere negatively with the antitumoractivity of Dx, either in vivo or in vitro. Micromolar concentrations of DPDP but not MnDPDP displayed anin vitro cytotoxic activity against A2780 cells. The present results show that MnDPDP, after being metabolized toMnPLED, protects against acute Dx cardiotoxicity. Both in vivo and in vitro experiments show that cardioprotectiontakes place without interfering negatively with the anticancer activity of Dx. Furthermore, the results suggest thatthe previously described cytotoxic in vivo activity of MnDPDP is an inherent property of DPDP.Translational Oncology (2012) 5, 252–259
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| 19. |
- Kurz, Tino, 1974-, et al.
(författare)
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Intralysosomal iron chelation protects against oxidative stress-induced cellular damage
- 2006
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 273:13, s. 3106-3117
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Tidskriftsartikel (refereegranskat)abstract
- Oxidant-induced cell damage may be initiated by peroxidative injury to lysosomal membranes, catalyzed by intralysosomal low mass iron that appears to comprise a major part of cellular redox-active iron. Resulting relocation of lytic enzymes and low mass iron would result in secondary harm to various cellular constituents. In an effort to further clarify this still controversial issue, we tested the protective effects of two potent iron chelators - the hydrophilic desferrioxamine (dfo) and the lipophilic salicylaldehyde isonicotinoyl hydrazone (sih), using cultured lysosome-rich macrophage-like J774 cells as targets. dfo slowly enters cells via endocytosis, while the lipophilic sih rapidly distributes throughout the cell. Following dfo treatment, long-term survival of cells cannot be investigated because dfo by itself, by remaining inside the lysosomal compartment, induces apoptosis that probably is due to iron starvation, while sih has no lasting toxic effects if the exposure time is limited. Following preincubation with 1 mm dfo for 3 h or 10 μm sih for a few minutes, both agents provided strong protection against an ensuing ∼LD50 oxidant challenge by preventing lysosomal rupture, ensuing loss of mitochondrial membrane potential, and apoptotic/necrotic cell death. It appears that once significant lysosomal rupture has occurred, the cell is irreversibly committed to death. The results lend strength to the concept that lysosomal membranes, normally exposed to redox-active iron in high concentrations, are initial targets of oxidant damage and support the idea that chelators selectively targeted to the lysosomal compartment may have therapeutic utility in diminishing oxidant-mediated cell injury. © 2006 The Authors.
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| 20. |
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| 21. |
- Kurz, Tino, 1974-, et al.
(författare)
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Lysosomal redox-active iron is important for oxidative stress-induced DNA damage
- 2004
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1019, s. 285-288
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Tidskriftsartikel (refereegranskat)abstract
- Data show that specifically chelating lysosomal redox-active iron can prevent most H2O2-induced DNA damage. Lysosomes seem to contain the major pool of redox-active labile iron within the cell. Under oxidative stress conditions, this iron may then relocate to the nucleus and play an important role for DNA damage by taking part in Fenton reactions.
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| 22. |
- Kurz, Tino, 1974-, et al.
(författare)
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Lysosomes and oxidative stress in aging and apoptosis
- 2008
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : Elsevier BV. - 0304-4165 .- 1872-8006. ; 1780:11, s. 1291-1303
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Tidskriftsartikel (refereegranskat)abstract
- The lysosomal compartment consists of numerous acidic vesicles (pH ~ 4-5) that constantly fuse and divide. It receives a large number of hydrolases from the trans-Golgi network, while their substrates arrive from both the cell's outside (heterophagy) and inside (autophagy). Many macromolecules under degradation inside lysosomes contain iron that, when released in labile form, makes lysosomes sensitive to oxidative stress. The magnitude of generated lysosomal destabilization determines if reparative autophagy, apoptosis, or necrosis will follow. Apart from being an essential turnover process, autophagy is also a mechanism for cells to repair inflicted damage, and to survive temporary starvation. The inevitable diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow oxidative formation of lipofuscin in long-lived postmitotic cells, where it finally occupies a substantial part of the volume of the lysosomal compartment. This seems to result in a misdirection of lysosomal enzymes away from autophagosomes, resulting in depressed autophagy and the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. This scenario might put aging into the category of autophagy disorders. © 2008 Elsevier B.V. All rights reserved.
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| 23. |
- Kurz, Tino, et al.
(författare)
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Lysosomes In Iron Metabolism, Ageing And Apoptosis
- 2008
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Ingår i: Histochemistry and Cell Biology. - : Institutionen för medicin och hälsa. - 0948-6143 .- 1432-119X. ; 129:4, s. 389-406
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Tidskriftsartikel (refereegranskat)abstract
- The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ~4-5) that constantly fuse and divide. It receives a large number of hydrolases (~50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while ‘resting’ lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.
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| 24. |
- Kurz, Tino, et al.
(författare)
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Oxidative Stress and Lysosomes
- 2011
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Ingår i: Principles of Free Radical Biomedicine. - : Nova Science Publishers, Inc.. - 9781612097749 ; , s. 1-18
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Bokkapitel (refereegranskat)abstract
- Recent years have witnessed an avalanche of new knowledge implicating free radicals in virtually every aspect of biology and medicine. It is now axiomatic that the regulated accumulation of reactive oxygen species (ROS) contributes to organismal health and well-being and that ROS serve as signaling molecules involved in cell growth, differentiation, gene regulation, replicative senescence and apoptosis. This book is an interdisciplinary text broken up into three consecutive volumes on the biochemistry and cellular/molecular biology of free radicals, transition metals, oxidants and antioxidants, and the role of oxidative stress in human health and disease.
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| 25. |
- Kurz, Tino, et al.
(författare)
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Redox Activity Within the Lysosomal Compartment: Implications for Aging and Apoptosis
- 2010
-
Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert, Inc.. - 1523-0864 .- 1557-7716. ; 13:4, s. 511-523
-
Forskningsöversikt (refereegranskat)abstract
- The lysosome is a redox-active compartment containing low-mass iron and copper liberated by autophagic degradation of metalloproteins. The acidic milieu and high concentration of thiols within lysosomes will keep iron in a reduced ( ferrous) state, which can react with endogenous or exogenous hydrogen peroxide. Consequent intralysosomal Fenton reactions may give rise to the formation of lipofuscin or "age pigment that accumulates in long-lived postmitotic cells that cannot dilute it by division. Extensive accumulation of lipofuscin seems to hinder normal autophagy and may be an important factor behind aging and age-related pathologies. Enhanced oxidative stress causes lysosomal membrane permeabilization, with ensuing relocation to the cytosol of iron and lysosomal hydrolytic enzymes, with resulting apoptosis or necrosis. Lysosomal copper is normally not redox active because it will form non-redox-active complexes with various thiols. However, if cells are exposed to lysosomotropic chelators that do not bind all the copper coordinates, highly redox-active complexes may form, with ensuing extensive lysosomal Fenton-type reactions and loss of lysosomal stability. Because many malignancies seem to have increased amounts of copper-containing macromolecules that are turned over by autophagy, it is conceivable that lysosomotropic copper chelators may be used in the future in ROS-based anticancer therapies.
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| 26. |
- Kurz, Tino, 1974-, et al.
(författare)
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Relocalized redox-active lysosomal iron is an important mediator of oxidative-stress-induced DNA damage
- 2004
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 378:3, s. 1039-1045
-
Tidskriftsartikel (refereegranskat)abstract
- Oxidative damage to nuclear DNA is known to involve site-specific Fenton-type chemistry catalysed by redox-active iron or copper in the immediate vicinity of DNA. However, the presence of transition metals in the nucleus has not been shown convincingly. Recently, it was proposed that a major part of the cellular pool of loose iron is confined within the acidic vacuolar compartment [Yu, Persson, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1243-1252, Persson, Yu, Tirosh, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1295-1305]. Consequently, rupture of secondary lysosomes, as well as subsequent relocation of labile iron to the nucleus, could be an important intermediary step in the generation of oxidative damage to DNA. To test this concept we employed the potent iron chelator DFO (desferrioxamine) conjugated with starch to form an HMM-DFO (high-molecular-mass DFO complex). The HMM-DFO complex will enter cells only via fluid-phase endocytosis and remain within the acidic vacuolar compartment, thereby chelating redox-active iron exclusively inside the endosomal/lysosomal compartment. Both free DFO and HMM-DFO equally protected lysosomal-membrane integrity against H2O 2-induced oxidative disruption. More importantly, both forms of DFO prevented H2O2-induced strand breaks in nuclear DNA, including telomeres. To exclude the possibility that lysosomal hydrolases, rather than iron, caused the observed DNA damage, limited lysosomal rupture was induced using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride, subsequently, hardly any DNA damage was found. These observations suggest that rapid oxidative damage to cellular DNA is minimal in the absence of redox-active iron and that oxidant-mediated DNA damage, observed in normal cells, is mainly derived from intralysosomal iron translocated to the nucleus after lysosomal rupture.
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| 27. |
- Kurz, Tino, et al.
(författare)
-
The role of lysosomes in iron metabolism and recycling
- 2011
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Ingår i: International Journal of Biochemistry and Cell Biology. - : Elsevier. - 1357-2725 .- 1878-5875. ; 43:12, s. 1686-1697
-
Forskningsöversikt (refereegranskat)abstract
- Iron is the most abundant transition metal in the earths crust. It cycles easily between ferric (oxidized; Fe(III)) and ferrous (reduced; Fe(II)) and readily forms complexes with oxygen, making this metal a central player in respiration and related redox processes. However, loose iron, not within heme or iron-sulfur cluster proteins, can be destructively redox-active, causing damage to almost all cellular components, killing both cells and organisms. This may explain why iron is so carefully handled by aerobic organisms. Iron uptake from the environment is carefully limited and carried out by specialized iron transport mechanisms. One reason that iron uptake is tightly controlled is that most organisms and cells cannot efficiently excrete excess iron. When even small amounts of intracellular free iron occur, most of it is safely stored in a non-redox-active form in ferritins. Within nucleated cells, iron is constantly being recycled from aged iron-rich organelles such as mitochondria and used for construction of new organelles. Much of this recycling occurs within the lysosome, an acidic digestive organelle. Because of this, most lysosomes contain relatively large amounts of redox-active iron and are therefore unusually susceptible to oxidant-mediated destabilization or rupture. In many cell types, iron transit through the lysosomal compartment can be remarkably brisk. However, conditions adversely affecting lysosomal iron handling (or oxidant stress) can contribute to a variety of acute and chronic diseases. These considerations make normal and abnormal lysosomal handling of iron central to the understanding and, perhaps, therapy of a wide range of diseases.
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| 28. |
- Peng, Xiang, et al.
(författare)
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The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca2+ signaling pathways
- 2014
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Ingår i: Thrombosis Research. - Oxon, United Kingdom : Elsevier. - 0049-3848 .- 1879-2472. ; 134:2, s. 418-425
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated.Methods: The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry.Results: PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca2+ mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca2+ chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it.Conclusion: The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca2+ signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.
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| 29. |
- Persson, H.Lennart, et al.
(författare)
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Radiation-induced cell death : Importance of lysosomal destabilization
- 2005
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 389:3, s. 877-884
-
Tidskriftsartikel (refereegranskat)abstract
- The mechanisms involved in radiation-induced cellular injury and death remain incompletely understood. In addition to the direct formation of highly reactive hydroxyl radicals (HO.) by radiolysis of water, oxidative stress events in the cytoplasm due to formation of H2O2 may also be important. Since the major pool of low-mass redox-active intracellular iron seems to reside within lysosomes, arising from the continuous intralysosomal autophagocytotic degradation of ferruginous materials, formation of H2O2 inside and outside these organelles may cause lysosomal labilization with release to the cytosol of lytic enzymes and low-mass iron. If of limited magnitude, such release may induce 'reparative autophagocytosis', causing additional accumulation of redox-active iron within the lysosomal compartment. We have used radio-resistant histiocytic lymphoma (J774) cells to assess the importance of intralysosomal iron and lysosomal rupture in radiation-induced cellular injury. We found that a 40 Gy radiation dose increased the 'loose' iron content of the (still viable) cells approx. 5-fold when assayed 24 h later. Cytochemical staining revealed that most redox-active iron was within the lysosomes. The increase of intralysosomal iron was associated with 'reparative autophagocytosis', and sensitized cells to Iysosomal rupture and consequent apoptotic/necrotic death following a second, much lower dose of radiation (20 Gy) 24 h after the first one. A high-molecular-mass derivative of desferrioxamine, which specifically localizes intralysosomally following endocytic uptake, added to the culture medium before either the first or the second dose of radiation, stabilized lysosomes and largely prevented cell death. These observations may provide a biological rationale for fractionated radiation. © 2005 Biochemical Society.
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| 30. |
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| 31. |
- Söderberg, Daniel, et al.
(författare)
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Increased levels of neutrophil extracellular trap remnants in the circulation of patients with small vessel vasculitis, but an inverse correlation to anti-neutrophil cytoplasmic antibodies during remission
- 2015
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Ingår i: Rheumatology. - : OXFORD UNIV PRESS. - 1462-0324 .- 1462-0332. ; 54:11, s. 2085-2094
-
Tidskriftsartikel (refereegranskat)abstract
- Objectives. Neutrophil extracellular traps (NETs) have been visualized at the site of ANCA-associated vasculitis (AAV) lesions. Increased levels of NET remnants in the circulation have been reported in some AAV patients with active disease. The aim of the present study was to analyse NET remnants in a larger cohort of AAV patients with varying degrees of disease activity and to elucidate possible factors responsible for remnant variation. Methods. Levels of NET remnants in the circulation of healthy controls (HCs; n =31) and AAV patients (n =93) were determined with ELISA. NET remnants were then correlated with ANCA levels, spontaneous and induced cell death (NETosis/necrosis) in vitro, neutrophil count and corticosteroid therapy. Results. Patients with active disease showed higher levels of circulating NET remnants compared with patients in remission (P=0.026) and HCs (P=0.006). From patients sampled during both remission and active disease, we found increased levels during active disease (P=0.0010). In remission, ANCA-negative patients had higher levels of NET remnants than ANCA-positive patients and a negative correlation was observed between NET remnants and PR3-ANCA (rs = 0.287, P=0.048). NET remnants correlated with neutrophil count in HCs (rs =0.503, P=0.014) but not in patients during remission. Neutrophils from patients showed enhanced spontaneous cell death (P=0.043). Conclusion. We found increased levels of circulating NET remnants in patients with active AAV. Furthermore, AAV patients exhibited an increased propensity for spontaneous cell death. NET remnant levels seem to be positively related to disease activity and neutrophil count, but inversely related to ANCA at least during remission.
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| 32. |
- Tenopoulou, Margarita, et al.
(författare)
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Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?
- 2007
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 403:2, s. 261-266
-
Tidskriftsartikel (refereegranskat)abstract
- The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at < pH 5. In the present study we show that the calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron. © 2007 Biochemical Society.
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| 33. |
- Terman, Alexei, et al.
(författare)
-
Lysosomal iron, iron chelation, and cell death
- 2013
-
Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert. - 1523-0864 .- 1557-7716. ; 18:8, s. 888-898
-
Forskningsöversikt (refereegranskat)abstract
- Significance: Lysosomes are acidic organelles containing more than fifty hydrolases that provide for the degradation of intracellular and endocytosed materials by autophagy and heterophagy, respectively. They digest a variety of macromolecules, as well as all organelles, and their integrity is crucial. As a result of the degradation of iron-containing macromolecules (e.g., ferritin and mitochondrial components) or endocytosed erythrocytes (by macrophages), lysosomes can accumulate large amounts of iron. This iron occurs often as Fe(II) due to the acidic and reducing lysosomal environment. Fe(II) is known to catalyze Fenton reactions, yielding extremely reactive hydroxyl radicals that may jeopardize lysosomal membrane integrity during oxidative stress. This results in the release of hydrolases and redox-active iron into the cytosol with ensuing damage or cell death. Lysosomes play key roles not only in apoptosis and necrosis but also in neurodegeneration, aging, and atherosclerosis. Recent Advances: The damaging effect of intralysosomal iron can be hampered by endogenous or exogenous iron chelators that enter the lysosomal compartment by membrane permeation, endocytosis, or autophagy. Critical Issues: Cellular sensitivity to oxidative stress is enhanced by lysosomal redox-active iron or by lysosomal-targeted copper chelators binding copper (from degradation of copper-containing macromolecules) in redox-active complexes. Probably due to higher copper levels, lysosomes of malignant cells may be specifically sensitized by such chelators. Future Directions: By increasing lysosomal redox-active iron or exposing cells to lysosomal-targeted copper chelators, it should be possible to enhance the sensitivity of cancer cells to radiation-induced oxidative stress or treatment with cytostatics that induce such stress.
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| 34. |
- Terman, Alexei, et al.
(författare)
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Lysosomal labilization
- 2006
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Ingår i: IUBMB Life - A Journal of the International Union of Biochemistry and Molecular Biology. - : Wiley. - 1521-6543 .- 1521-6551. ; 58:9, s. 531-539
-
Forskningsöversikt (refereegranskat)abstract
- The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells. © 2006 IUBMB.
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| 35. |
- Terman, Alexei, et al.
(författare)
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Mitochondrial Turnover and Aging of Long-Lived Postmitotic Cells: The Mitochondrial-Lysosomal Axis Theory of Aging
- 2010
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Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert Inc. - 1523-0864 .- 1557-7716. ; 12:4, s. 503-535
-
Forskningsöversikt (refereegranskat)abstract
- It is now generally accepted that aging and eventual death of multicellular organisms is to a large extent related to macromolecular damage by mitochondrially produced reactive oxygen species, mostly affecting long-lived postmitotic cells, such as neurons and cardiac myocytes. These cells are rarely or not at all replaced during life and can be as old as the whole organism. The inherent inability of autophagy and other cellular-degradation mechanisms to remove damaged structures completely results in the progressive accumulation of garbage, including cytosolic protein aggregates, defective mitochondria, and lipofuscin, an intralysosomal indigestible material. In this review, we stress the importance of crosstalk between mitochondria and lysosomes in aging. The slow accumulation of lipofuscin within lysosomes seems to depress autophagy, resulting in reduced turnover of effective mitochondria. The latter not only are functionally deficient but also produce increased amounts of reactive oxygen species, prompting lipofuscinogenesis. Moreover, defective and enlarged mitochondria are poorly autophagocytosed and constitute a growing population of badly functioning organelles that do not fuse and exchange their contents with normal mitochondria. The progress of these changes seems to result in enhanced oxidative stress, decreased ATP production, and collapse of the cellular catabolic machinery, which eventually is incompatible with survival.
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| 36. |
- Terman, Alexei, et al.
(författare)
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The involvement of lysosomes in myocardial aging and disease
- 2008
-
Ingår i: Current Cardiology Reviews. - Bussum, Netherlands : Bentham Science Publishers Ltd.. - 1573-403X. ; 4:2, s. 107-115
-
Forskningsöversikt (refereegranskat)abstract
- The myocardium is mainly composed of long-lived postmitotic cells with, if there is any at all, a very low rate of replacement through the division and differentiation of stem cells. As a consequence, cardiac myocytes gradually undergo pronounced age-related alterations which, furthermore, occur at a rate that inversely correlates with the longevity of species. Basically, these alterations represent the accumulation of structures that have been damaged by oxidation and that are useless and often harmful. These structures (so-called 'waste' materials), include defective mitochondria, aberrant cytosolic proteins, often in aggregated form, and lipofuscin, which is an intralysosomal undegradable polymeric substance. The accumulation of 'waste' reflects the insufficient capacity for autophagy of the lysosomal compartment, as well, as the less than perfect functioning of proteasomes, calpains and other cellular digestive systems. Senescent mitochondria are usually enlarged, show reduced potential over their inner membrane, are deficient in ATP production, and often produce increased amounts of reactive oxygen species. The turnover of damaged cellular structures is hindered by an increased lipofuscin loading of the lysosomal compartment. This particularly restricts the autophagic turnover of enlarged, defective mitochondria, by diverting the flow of lysosomal hydrolases from autophagic vacuoles to lipofuscin-loaded lysosomes where the enzymes are lost, since lipofuscin is not degradable by lysosomal hydrolases. As a consequence, aged lipofuscin-rich cardiac myocytes become overloaded with damaged mitochondria, leading to increased oxidative stress, apoptotic cell death, and the gradual development of heart failure. Defective lysosomal function also underlies myocardial degeneration in various lysosomal storage diseases, while other forms of cardiomyopathies develop due to mitochondrial DNA mutations, resulting in an accumulation of abnormal mitochondria that are not properly eliminated by autophagy. The degradation of iron-saturated ferritin in lysosomes mediates myocardial injury in hemochromatosis, an acquired or hereditary disease associated with iron overload. Lysosomes then become sensitized to oxidative stress by the overload of low mass, redox-active iron that accumulates when iron-saturated ferritin is degraded following autophagy. Lysosomal destabilization is of importance in the induction and/or execution of programmed cell death (either classical apoptotic or autophagic), which is a common manifestation of myocardial aging and a variety of cardiac pathologies. © 2008 Bentham Science Publishers Ltd.
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| 37. |
- Ungvari, Zoltan, et al.
(författare)
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Resistance to Genotoxic Stresses in Arctica islandica, the Longest Living Noncolonial Animal : Is Extreme Longevity Associated With a Multistress Resistance Phenotype?
- 2013
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Ingår i: The journals of gerontology. Series A, Biological sciences and medical sciences. - : Oxford University Press. - 1079-5006 .- 1758-535X. ; 68:5, s. 521-529
-
Tidskriftsartikel (refereegranskat)abstract
- Bivalve molluscs are newly discovered models of successful aging. Here, we test the hypothesis that extremely long-lived bivalves are not uniquely resistant to oxidative stressors (eg, tert-butyl hydroperoxide, as demonstrated in previous studies) but exhibit a multistress resistance phenotype. We contrasted resistance (in terms of organismal mortality) to genotoxic stresses (including topoisomerase inhibitors, agents that cross-link DNA or impair genomic integrity through DNA alkylation or methylation) and to mitochondrial oxidative stressors in three bivalve mollusc species with dramatically differing life spans: Arctica islandica (ocean quahog), Mercenaria mercenaria (northern quahog), and the Atlantic bay scallop, Argopecten irradians irradians (maximum species life spans: >500, >100, and ~2 years, respectively). With all stressors, the short-lived A i irradians were significantly less resistant than the two longer lived species. Arctica islandica were consistently more resistant than M mercenaria to mortality induced by oxidative stressors as well as DNA methylating agent nitrogen mustard and the DNA alkylating agent methyl methanesulfonate. The same trend was not observed for genotoxic agents that act through cross-linking DNA. In contrast, M mercenaria tended to be more resistant to epirubicin and genotoxic stressors, which cause DNA damage by inhibiting topoisomerases. To our knowledge, this is the first study comparing resistance to genotoxic stressors in bivalve mollusc species with disparate longevities. In line with previous studies of comparative stress resistance and longevity, our data extends, at least in part, the evidence for the hypothesis that an association exists between longevity and a general resistance to multiplex stressors, not solely oxidative stress. This work also provides justification for further investigation into the interspecies differences in stress response signatures induced by a diverse array of stressors in short-lived and long-lived bivalves, including pharmacological agents that elicit endoplasmic reticulum stress and cellular stress caused by activation of innate immunity.
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