32 results on '"Goralska M"'
Search Results
2. Ceruloplasmin alters intracellular iron regulated proteins and pathways: Ferritin, transferrin receptor, glutamate and hypoxia-inducible factor-1α
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Harned, J., Ferrell, J., Nagar, S., Goralska, M., Fleisher, L. N., and McGahan, M. C.
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- 2012
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3. Distribution of ferritin chains in canine lenses with and without age-related nuclear cataracts
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Goralska, M., Nagar, S., Fleisher, L.N., and McGahan, M.C.
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Aging ,genetic structures ,Iron ,Blotting, Western ,Lens Nucleus, Crystalline ,eye diseases ,Cataract ,Molecular Weight ,Dogs ,Apoferritins ,Animals ,Electrophoresis, Polyacrylamide Gel ,sense organs ,Dog Diseases ,Research Article - Abstract
Purpose It was determined in an earlier study that ferritin-heavy (H) and -light (L) chains in lens fiber cells are modified in comparison to those in lens epithelial cells. The purpose of the present study was to determine whether changes in ferritin chain characteristics are developmental, age-related, or associated with cataractogenesis, by analyzing the distribution of modified chains throughout the lens fiber mass. Methods After removing the capsule, noncataractous and cataractous lenses were separated into six layers of fiber cells. The content of ferritin H and L chains in each layer was determined by western blotting with chain-specific antibodies. The level of ferritin complex (450 kDa protein made up of assembled L and H chains) was determined using the enzyme-linked immunosorbent assay. The ability of ferritin complex to bind iron was assessed by in vitro labeling with 59Fe. Results Fiber cell ferritin L chains were 30 kDa (modified from the normal 19 kDa), and were present at the highest level in the outermost layers of both cataractous and non-cataractous lenses. The amount of modified L chains decreased gradually in the inner layers of the fiber mass, and was undetectable in the inner two layers of cataractous lenses. The ferritin H chains were also modified to 12 kDa (perhaps truncated from the normal 21 kDa size) in both cataractous and non-cataractous lenses. Similar levels of this modified H chain were found throughout the normal lens. Interestingly, in cataractous lenses, the modified H chains were found in decreasing amounts towards the interior of the lens, and were undetectable in the nucleus. However, in these cataractous lenses, the normal-sized ferritin H chains (21 kDA) appear in small quantities in the outer fiber layers, and increase in quantity and size (to 29 kDa) in the inner layers. This observation was best seen and demonstrated in advanced cataracts. Ferritin, which can bind iron, was found mainly in the outer layers of the lens fiber mass of normal lenses, but was more evenly distributed in fiber layers from cataractous lenses. Conclusions Both ferritin H and L chains were modified in lens fiber cells from normal and cataractous canine lenses. These modifications were not age-related, and most likely occur during the differentiation of epithelial cells to fiber cells, since only normal-sized chains have been found in lens epithelial cells. In addition, there was a specific and distinct distribution of these modified chains throughout the lens fiber mass. The most striking differences between normal and cataractous lenses fiber cells were the appearance of normal-sized ferritin H chains and the relatively even distribution of iron binding capacity throughout the fiber mass of the cataractous lenses. These differences may reflect a response of the lens to increased oxidative stress during cataractogenesis.
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- 2009
4. The effects of Tempol on ferritin synthesis and Fe metabolism in lens epithelial cells
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Goralska, M, primary, Holley, B, additional, and McGahan, M.C, additional
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- 2000
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5. The Effect of Ascorbic Acid and Ferric Ammonium Citrate on Iron Uptake and Storage in Lens Epithelial Cells
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GORALSKA, M., primary, HARNED, J., additional, FLEISHER, L.N., additional, and McGAHAN, M.C., additional
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- 1998
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6. Mechanisms by which Ascorbic Acid Increases Ferritin levels in Cultured Lens Epithelial Cells
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GORALSKA, M., primary, HARNED, J., additional, GRIMES, A.M., additional, FLEISHER, L.N., additional, and McGAHAN, M.C., additional
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- 1997
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7. Transferrin secretion by lens epithelial cells in culture
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McGahan, M.C., primary, Harned, J., additional, Goralska, M., additional, Sherry, B., additional, and Fleisher, L.N., additional
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- 1995
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8. Some behavioural effects of antidepressant drugs are time-dependent
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Dziedzicka-Wasylewska, M., Rogoz, Z., Margas, W., Dlaboga, D., and Goralska, M.
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- 2001
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9. Kynuramines induce overexpression of heat shock proteins in pancreatic cancer cells Via 5-hydroxytryptamine and MT1/MT2 receptors
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Leja-Szpak, A., Piotr Pierzchalski, Goralska, M., Nawrot-Porabka, K., Bonior, J., Link-Lenczowski, P., Jastrzebska, M., and Jaworek, J.
10. Melatonin metabolite, N1-acetyl-N1-formyl-5-methoxykynuramine (AFMK), attenuates acute pancreatitis in the rat: In vivo and in vitro studies
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Jaworek, J., Szklarczyk, J., Joanna Bonior, Kot, M., Goralska, M., Pierzchalski, P., Reiter, R. J., Czech, U., and Tomaszewska, R.
11. The effect of antidepressant drugs on transcriptional activity of dopamine D 2 receptor gene promoter in the NB41A3 cells — In vitro study
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Góralska, M., Kucharska, A., Sobota, R., and Dziedzicka-Wasylewska, M.
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- 2000
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12. The effect of antidepressant drugs on transcriptional activity of dopamine D2 receptor gene promoter in the NB41A3 cells - In vitro study
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Goralska, M., Kucharska, A., Sobota, R., Dziedzicka-Wasylewska, and M.
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- 2000
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13. Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells.
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Goralska M, Fleisher LN, and McGahan MC
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- Animals, Blotting, Western, Cells, Cultured, Disease Models, Animal, Dogs, Immunohistochemistry, Aquaporins metabolism, Epithelial Cells metabolism, Eye Proteins metabolism, Ferritins metabolism, Lens, Crystalline metabolism, Receptors, Transferrin metabolism, Vitreous Body physiology
- Abstract
Purpose: In humans, vitrectomy is associated with development of nuclear cataracts. Iron catalyzes free radical formation causing oxidative damage, which is implicated in cataract formation. This study was designed to determine if vitreous humor, which can initiate differentiation of lens epithelial cells, would have an effect on iron-handling proteins., Methods: Cultured canine lens epithelial cells were treated with collected canine vitreous humor. Lysates of treated and control cells were separated by SDS-PAGE. Ferritin H- and L-chains, transferrin receptor 1, and aquaporin 0 were immunodetected and quantitated with specific antibodies. Morphologic changes in treated cells were assessed., Results: Treatment of lens epithelial cells with a 33% (vol/vol) solution of vitreous humor changed the morphology of lens cells and induced expression of aquaporin 0, a marker of fiber cell differentiation that was undetectable in control cells. Treatment did not modify the size of iron-handling proteins but significantly increased content of ferritin from 2.9- to 8.8-fold over control and decreased levels of transferrin receptor by 37% to 59%., Conclusions: Vitreous humor may significantly limit iron uptake by transferrin/transferrin receptor pathway, and by increasing ferritin levels could profoundly increase the iron-storage capacity of ferritin in lens cells. Vitreous humor may play a significant protective role against iron-catalyzed oxidative damage of lens epithelial cells and therefore in the formation of cataracts.
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- 2017
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14. Melatonin metabolite, N(1)-acetyl-N(1)-formyl-5-methoxykynuramine (AFMK), attenuates acute pancreatitis in the rat: in vivo and in vitro studies.
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Jaworek J, Szklarczyk J, Bonior J, Kot M, Goralska M, Pierzchalski P, Reiter RJ, Czech U, and Tomaszewska R
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- Acute Disease, Aldehydes metabolism, Amylases blood, Animals, Anti-Inflammatory Agents pharmacology, Antioxidants therapeutic use, Caspase 3 metabolism, Cell Line, Tumor, Glutathione Peroxidase metabolism, Kynuramine pharmacology, Kynuramine therapeutic use, Male, Malondialdehyde metabolism, Melatonin metabolism, Pancreas drug effects, Pancreas metabolism, Pancreas pathology, Pancreatitis blood, Pancreatitis metabolism, Pancreatitis pathology, Proto-Oncogene Proteins c-bcl-2 metabolism, Rats, Wistar, Tumor Necrosis Factor-alpha blood, Tumor Necrosis Factor-alpha metabolism, bcl-2-Associated X Protein metabolism, Anti-Inflammatory Agents therapeutic use, Kynuramine analogs & derivatives, Pancreatitis drug therapy
- Abstract
Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 μg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.
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- 2016
15. Kynuramines induce overexpression of heat shock proteins in pancreatic cancer cells via 5-hydroxytryptamine and MT1/MT2 receptors.
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Leja-Szpak A, Pierzchalski P, Goralska M, Nawrot-Porabka K, Bonior J, Link-Lenczowski P, Jastrzebska M, and Jaworek J
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- Cell Line, Tumor, Humans, Ketanserin pharmacology, Melatonin metabolism, Receptor, Melatonin, MT1, Receptor, Melatonin, MT2 metabolism, Tropanes pharmacology, Tryptamines pharmacology, Tryptophan metabolism, Heat-Shock Proteins metabolism, Kynuramine metabolism, Pancreatic Neoplasms metabolism, Serotonin metabolism
- Abstract
Kynuramines, metabolites of melatonin and L-tryptophan, are synthesized endogenously by oxygenases or in spontaneous reaction by an interaction with free radicals. We have reported previously that melatonin stimulates expression and phosphorylation of heat shock protein (HSP) 27, as well as production of HSP70 and HSP90αβ in pancreatic carcinoma cells (PANC-1). Based on those results, we hypothesized that above processes could have been involved in the interruption of intrinsic proapoptotic pathway. Herein, we report that incubation of PANC-1 cells with N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) or with L-kynurenine (L-KYN) lead to the overexpression of heat shock protein synthesis and these effects are partially reversed by 5-HT3 or MT1/MT2 receptor antagonists. PANC-1 cells in culture were treated with AFMK or L-KYN, with non selective MT1/MT2 receptor antagonist (luzindole), with 5-HT2 and 5-HT3 receptor antagonists (ketanserin and MDL72222), or combination of these substances. Both AFMK and L-KYN significantly decreased cytoplasmic HSP27 and this effect was presumably due to increased of its phosphorylation and consequent nuclear translocation, confirmed by immunoprecipitation of phosphorylated form of HSP27. These changes were accompanied by marked augmentation of HSP70 and HSP90αβ in the cytosolic fraction. Pretreatment of cell cultures with luzindole or MDL72222 followed by the addition of AFMK or L-KYN reversed the stimulatory effects of these substances on HSP expression in PANC-1 cells, whereas ketanserin failed to influence mentioned above phenomenon. We conclude that activation of HSPs in pancreatic carcinoma cells seems to be dependent on an interaction of AFMK or L-KYN with MT1/MT2 or/and 5-HT3 receptors.
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- 2015
16. Hypoxia induced changes in expression of proteins involved in iron uptake and storage in cultured lens epithelial cells.
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Goralska M, Fleisher LN, and McGahan MC
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- Animals, Cells, Cultured, Dogs, Gene Expression Regulation, Lens, Crystalline cytology, Oxidative Stress physiology, RNA, Messenger metabolism, Real-Time Polymerase Chain Reaction, Cell Hypoxia physiology, Epithelial Cells metabolism, Ferritins metabolism, Lens, Crystalline metabolism, Receptors, Transferrin metabolism, Transferrin metabolism
- Abstract
Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides., (Copyright © 2014. Published by Elsevier Ltd.)
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- 2014
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17. Source-dependent intracellular distribution of iron in lens epithelial cells cultured under normoxic and hypoxic conditions.
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Goralska M, Nagar S, Fleisher LN, Mzyk P, and McGahan MC
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- Animals, Biological Transport, Cell Nucleus metabolism, Cell Proliferation, Cell Survival, Cells, Cultured, Dogs, Intracellular Fluid metabolism, Iron Radioisotopes metabolism, Lens, Crystalline metabolism, Mitochondria metabolism, Chlorides metabolism, Epithelial Cells metabolism, Ferric Compounds metabolism, Hypoxia metabolism, Lens, Crystalline cytology, Transferrin metabolism
- Abstract
Purpose: Intracellular iron trafficking and the characteristics of iron distribution from different sources are poorly understood. We previously determined that the lens removes excess iron from fluids of inflamed eyes. In the current study, we examined uptake and intracellular distribution of ⁵⁹Fe from iron transport protein transferrin or ferric chloride (nontransferrin-bound iron [NTBI]) in cultured canine lens epithelial cells (LECs). Because lens tissue physiologically functions under low oxygen tension, we also tested effects of hypoxia on iron trafficking. Excess iron, not bound to proteins, can be damaging to cells due to its ability to catalyze formation of reactive oxygen species., Methods: LECs were labeled with ⁵⁹Fe-Tf or ⁵⁹FeCl₃ under normoxic or hypoxic conditions. Cell lysates were fractioned into mitochondria-rich, nuclei-rich, and cytosolic fractions. Iron uptake and its subcellular distribution were measured by gamma counting., Results: ⁵⁹Fe accumulation into LECs labeled with ⁵⁹Fe-Tf was 55-fold lower as compared with that of ⁵⁹FeCl₃. Hypoxia (24 hours) decreased uptake of iron from transferrin but not from FeCl₃. More iron from ⁵⁹FeCl₃ was directed to the mitochondria-rich fraction (32.6%-47.7%) compared with ⁵⁹Fe from transferrin (10.6%-12.6%). The opposite was found for the cytosolic fraction (8.7%-18.3% and 54.2%-46.6 %, respectively). Hypoxia significantly decreased iron accumulation in the mitochondria-rich fraction of LECs labeled with ⁵⁹Fe-Tf ., Conclusions: There are source-dependent differences in iron uptake and trafficking. Uptake and distribution of NTBI are not as strictly regulated as that of iron from transferrin. Excessive exposure to NTBI, which could occur in pathological conditions, may oxidatively damage organelles, particularly mitochondria.
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- 2013
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18. Altered ferritin subunit composition: change in iron metabolism in lens epithelial cells and downstream effects on glutathione levels and VEGF secretion.
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Harned J, Ferrell J, Lall MM, Fleisher LN, Nagar S, Goralska M, and McGahan MC
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- Animals, Apoferritins genetics, Cell Nucleus metabolism, Cells, Cultured, Culture Media, Conditioned pharmacology, Cystine pharmacokinetics, Dogs, Epithelial Cells cytology, Homeostasis physiology, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Lens, Crystalline cytology, Oxidative Stress physiology, Protein Subunits genetics, Protein Subunits metabolism, RNA, Small Interfering, Transfection, Apoferritins metabolism, Epithelial Cells metabolism, Glutathione metabolism, Iron metabolism, Lens, Crystalline metabolism, Vascular Endothelial Growth Factor A metabolism
- Abstract
Purpose: The iron storage protein ferritin is necessary for the safe storage of iron and for protection against the production of iron-catalyzed oxidative damage. Ferritin is composed of 24 subunits of two types: heavy (H) and light (L). The ratio of these subunits is tissue specific, and alteration of this ratio can have profound effects on iron storage and availability. In the present study, siRNA for each of the chains was used to alter the ferritin H:L chain ratio and to determine the effect of these changes on ferritin synthesis, iron metabolism, and downstream effects on iron-responsive pathways in canine lens epithelial cells., Methods: Primary cultures of canine lens epithelial cells were used. The cells were transfected with custom-made siRNA for canine ferritin H- and L-chains. De novo ferritin synthesis was determined by labeling newly synthesized ferritin chains with 35S-methionine, immunoprecipitation, and separation by SDS-PAGE. Iron uptake into cells and incorporation into ferritin was measured by incubating the cells with 59Fe-labeled transferrin. Western blot analysis was used to determine the presence of transferrin receptor, and ELISA was used to determine total ferritin concentration. Ferritin localization in the cells was determined by immunofluorescence labeling. VEGF, glutathione secretion levels, and cystine uptake were measured., Results: FHsiRNA decreased ferritin H-chain synthesis, but doubled ferritin L-chain synthesis. FLsiRNA decreased both ferritin H- and L-chain synthesis. The degradation of ferritin H-chain was blocked by both siRNAs, whereas only FHsiRNA blocked the degradation of ferritin L-chain, which caused significant accumulation of ferritin L-chain in the cells. This excess ferritin L-chain was found in inclusion bodies, some of which were co-localized with lysosomes. Iron storage in ferritin was greatly reduced by FHsiRNA, resulting in increased iron availability, as noted by a decrease in transferrin receptor levels and iron uptake from transferrin. Increased iron availability also increased cystine uptake and glutathione concentration and decreased nuclear translocation of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor (VEGF) accumulation in the cell-conditioned medium., Conclusions: Most of the effects of altering the ferritin H:L ratio with the specific siRNAs were due to changes in the availability of iron in a labile pool. They caused significant changes in iron uptake and storage, the rate of ferritin synthesis and degradation, the secretion of VEGF, and the levels of glutathione in cultured lens epithelial cells. These profound effects clearly demonstrate that maintenance of a specific H:L ratio is part of a basic cellular homeostatic mechanism.
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- 2010
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19. Changes in ferritin H- and L-chains in canine lenses with age-related nuclear cataract.
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Goralska M, Nagar S, Colitz CM, Fleisher LN, and McGahan MC
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- Animals, Cataract metabolism, Dogs, Electrophoresis, Polyacrylamide Gel veterinary, Enzyme-Linked Immunosorbent Assay veterinary, Fluorescent Antibody Technique, Indirect veterinary, Microscopy, Fluorescence veterinary, Aging, Apoferritins metabolism, Cataract veterinary, Dog Diseases metabolism, Lens, Crystalline metabolism
- Abstract
Purpose: To determine potential differences in the characteristics of the iron storage protein ferritin and its heavy (H) and light (L) subunits in fiber cells from cataractous and noncataractous lenses of older dogs., Methods: Lens fiber cell homogenates were analyzed by SDS-PAGE, and ferritin chains were immunodetected with ferritin chain-specific antibodies. Ferritin concentration was measured by ELISA. Immunohistochemistry was used to localize ferritin chains in lens sections., Results: The concentration of assembled ferritin was comparable in noncataractous and cataractous lenses of similarly aged dogs. The ferritin L-chain detected in both lens types was modified and was approximately 11 kDa larger (30 kDa) than standard L-chain (19 kDa) purified from canine liver. The H-chain identified in cataractous fiber cells (29 kDa) differed from the 21-kDa standard canine H-chain and from the 12-kDa modified H-chain present in fiber cells of noncataractous lenses. Histologic analysis revealed that the H-chain was distributed differently throughout cataractous lenses compared with noncataractous lenses. There was also a difference in subunit makeup of assembled ferritin between the two lens types. Ferritin from cataractous lenses contained more H-chain and bound 11-fold more iron than ferritin from noncataractous lenses., Conclusions: There are significant differences in the characteristics of ferritin H-chain and its distribution in canine cataractous lenses compared with noncataractous lenses. The higher content of H-chain in assembled ferritin allows this molecule to sequester more iron. In addition, the accumulation of H-chain in deeper fiber layers of the lens may be part of a defense mechanism by which the cataractous lens limits iron-catalyzed oxidative damage.
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- 2009
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20. Ferritin H- and L-chains in fiber cell canine and human lenses of different ages.
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Goralska M, Fleisher LN, and McGahan MC
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- Adolescent, Adult, Aged, Animals, Blotting, Western, Dogs, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Humans, Iron Radioisotopes metabolism, Lens, Crystalline cytology, Middle Aged, Aging physiology, Apoferritins metabolism, Ferritins metabolism, Lens, Crystalline metabolism
- Abstract
Purpose: This study was designed to elucidate potential age-related changes in the concentration, structure, and assembly pattern of ferritin chains in lens fiber cells., Methods: Canine and human lens fiber cell homogenate proteins were separated by one-dimensional and two-dimensional SDS-PAGE. Ferritin chains were immunodetected and quantitated with ferritin chain-specific antibodies. Total ferritin concentration was measured by ELISA. Binding of iron was determined in vitro with (59)Fe., Results: Ferritin H- and L-chains in canine and human fiber cells of healthy lenses were extensively modified. The H-chain in both species was truncated, and its concentration increased with age. Canine L-chain was approximately 11 kDa larger than standard canine L-chain, whereas human L-chain was of the proper size. Two-dimensional separation revealed age-related polymorphism of human and canine lens fiber cell L-chains and human H-chains. Normal size ferritin chains were not identified in canine fiber cells, but a small amount of fully assembled ferritin was detected, and its concentration decreased with age., Conclusions: Such significantly altered ferritin chains are not likely to form functional ferritin capable of storing iron. Therefore, lens fiber cells, particularly from older lenses, may have limited ability to protect themselves against iron-catalyzed oxidative damage.
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- 2007
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21. Differential degradation of ferritin H- and L-chains: accumulation of L-chain-rich ferritin in lens epithelial cells.
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Goralska M, Nagar S, Fleisher LN, and McGahan MC
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- Aging physiology, Animals, Apoferritins, Cells, Cultured, Cysteine Proteinase Inhibitors pharmacology, Dogs, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Epithelial Cells drug effects, Iron metabolism, Lens, Crystalline drug effects, Lysosomes metabolism, Methionine metabolism, Plasmids, Transfection, Epithelial Cells metabolism, Ferritins metabolism, Lens, Crystalline metabolism
- Abstract
Purpose: The storage of iron by ferritin is determined by tissue-specific composition of its 24 subunits, which are designated as either heavy (H) or light (L). For a better understanding of how lens epithelial cells regulate their ferritin subunit makeup, the degradation pattern of each subunit type was analyzed. In addition, age-related changes in ferritin concentration and subunit makeup were determined., Methods: Ferritin turnover in primary cultures of canine lens epithelial cells was determined by metabolic labeling with [(35)S]-methionine. Transient transfection with vectors containing coding sequences for either H- or L-chains were used to modify ferritin subunit makeup. Ferritin concentration was measured by ELISA. Immunodetection and fluorescence immunocytochemistry were used to study age-related changes in ferritin chain concentration., Results: Inhibition of the proteasomal protein degradation pathway by clastolactacystin-beta-lactone had no effect on ferritin degradation, whereas inhibition of lysosomal degradation markedly increased ferritin levels, confirming that this system is involved in ferritin turnover. H-chain ferritin degraded at a faster rate than the L-chain. L-chain-rich ferritin in L-chain-transfected cells formed inclusion bodies that were localized to the cytosol. Similar inclusion bodies were found in older lens cells kept in cell culture for more than 8 days., Conclusions: Steady degradation of H-chain ferritin contributed to the maintenance of a constant level of this chain within the lens epithelial cells. In contrast, slower turnover of the L-chain resulted in accumulation of L-chain-enriched ferritin associated with cytoplasmic inclusion bodies. These L-chain-containing inclusion bodies were found in the cytosol of cells overexpressing L-ferritin chain and in nontransfected cells maintained in culture for 8 to 35 days. Overexpression of the L-chain has been associated with the formation of premature cataracts in humans with hereditary hyperferritinemia cataract syndrome. The formation of inclusion bodies in older lens epithelial cells, as demonstrated in the current investigation, is intriguing and could point to possible involvement of cytoplasmic L-chain-enriched ferritin aggregates in the formation of age-related cataract.
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- 2005
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22. Iron alters glutamate secretion by regulating cytosolic aconitase activity.
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McGahan MC, Harned J, Mukunnemkeril M, Goralska M, Fleisher L, and Ferrell JB
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- Aconitate Hydratase antagonists & inhibitors, Animals, Cells, Cultured, Citric Acid metabolism, Dogs, Enzyme Activation drug effects, Isocitrates metabolism, Membrane Transport Proteins metabolism, Oxalates pharmacology, Pigment Epithelium of Eye drug effects, Riluzole pharmacology, Vesicular Glutamate Transport Protein 1, Aconitate Hydratase metabolism, Cytosol metabolism, Glutamates metabolism, Iron metabolism, Pigment Epithelium of Eye enzymology
- Abstract
Glutamate has many important physiological functions, including its role as a neurotransmitter in the retina and the central nervous system. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and that this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells, indicating that this may be a common mechanism for the regulation of glutamate production in many cell types. The activity of the iron-dependent enzyme cytosolic aconitase (c-aconitase) is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three-step process leading to glutamate formation. In the present study, iron increased c-aconitase activity, and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron-induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and retinal degeneration. Our results are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in the regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many nonneuronal tissues. We provide the first evidence that, in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter VGLUT1 and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole.
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- 2005
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23. Identification of a mechanism by which lens epithelial cells limit accumulation of overexpressed ferritin H-chain.
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Goralska M, Holley BL, and McGahan MC
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- Animals, Apoferritins, Blotting, Western, Cataract metabolism, Cytosol metabolism, DNA, Complementary metabolism, Dogs, Electrophoresis, Polyacrylamide Gel, Eye Diseases, Hereditary metabolism, Ferritins chemistry, Ferritins metabolism, Humans, Iron Metabolism Disorders, Mutation, Peptides chemistry, Plasmids metabolism, Precipitin Tests, RNA, Messenger metabolism, Recombinant Proteins metabolism, Time Factors, Transfection, Epithelial Cells metabolism, Ferritins biosynthesis, Lens, Crystalline metabolism
- Abstract
The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.
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- 2003
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24. Alpha lipoic acid changes iron uptake and storage in lens epithelial cells.
- Author
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Goralska M, Dackor R, Holley B, and McGahan MC
- Subjects
- Animals, Ascorbic Acid pharmacology, Cells, Cultured, Dogs, Dose-Response Relationship, Drug, Epithelial Cells metabolism, Ferritins biosynthesis, Ferritins metabolism, Hydrogen Peroxide antagonists & inhibitors, Hydrogen Peroxide pharmacology, Lens Capsule, Crystalline cytology, Lens Capsule, Crystalline metabolism, Oxidative Stress drug effects, Antioxidants pharmacology, Epithelial Cells drug effects, Iron pharmacokinetics, Lens Capsule, Crystalline drug effects, Thioctic Acid pharmacology
- Abstract
Alpha lipoic acid (LA) is a cofactor in mitochondrial dehydrogenase complexes. Previous studies have shown that when administered exogenously LA has antioxidant properties, which include free radical scavenging, metal chelation and regeneration of other antioxidants. The cells convert LA into dihydroplipoic acid (DHLA), which in the presence of iron can act as a prooxidant. In vitro DHLA reduces Fe(+3) to Fe(+2) and removes iron from ferritin, increasing the risk of Fe catalyzed free radical formation. In the present study we examined the in vivo effects of lipoic acid treatment on Fe metabolism in cultured lens epithelial cells, and found that LA decreases Fe uptake from transferrin, increases Fe deposition into ferritin and increases the concentration of this protein. When administered together with ascorbic acid, lipoic acid changes the characteristic heavy to light chain ratio of ferritin makeup. The decreased Fe uptake and increased storage diminishes the size of the cytosolic highly reactive Fe pool (LIP). These changes are associated with increased cell resistance to H(2)O(2) challenge. Therefore, LA may reduce the risk of Fe induced oxidative damage and also might be useful as a treatment of Fe overload., (Copyright 2003 Elsevier Science Ltd.)
- Published
- 2003
- Full Text
- View/download PDF
25. Overexpression of H- and L-ferritin subunits in lens epithelial cells: Fe metabolism and cellular response to UVB irradiation.
- Author
-
Goralska M, Holley BL, and McGahan MC
- Subjects
- Amino Acid Sequence, Animals, Apoferritins, Base Sequence, Cell Count, Cells, Cultured, DNA, Complementary metabolism, Dogs, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Epithelial Cells metabolism, Ferritins genetics, Gene Expression, Genetic Vectors, Humans, Lens, Crystalline metabolism, Molecular Sequence Data, Oxidative Stress, Transfection, Ultraviolet Rays, Epithelial Cells radiation effects, Ferritins biosynthesis, Iron metabolism, Lens, Crystalline radiation effects
- Abstract
Purpose: To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress., Methods: Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA: Fe uptake and incorporation into ferritin was determined by incubating transfected cells with (59)Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation., Results: Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected., Conclusions: Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.
- Published
- 2001
26. Basic fibroblast growth factor in fibrosing alveolitis induced by oxygen stress.
- Author
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Sannes PL, Khosla J, Johnson S, Goralska M, McGahan C, and Menard M
- Subjects
- Animals, Blotting, Northern, Rats, Rats, Sprague-Dawley, Fibroblast Growth Factor 2 metabolism, Oxygen pharmacology, Pulmonary Alveoli metabolism, Pulmonary Fibrosis metabolism
- Published
- 1996
- Full Text
- View/download PDF
27. Isolation of the fraction inhibiting erythropoiesis from the plasma of animals with experimental polycythemia.
- Author
-
Krzymowski T, Goralska M, and Ziecik A
- Subjects
- Animals, Female, Male, Mice, Sheep, Erythropoiesis, Polycythemia blood
- Published
- 1974
28. Plasma proteins in the period of posttransfusion polycythaemia in rats.
- Author
-
Goralska M and Ruszczyńska H
- Subjects
- Alpha-Globulins analysis, Animals, Blood Transfusion, Erythropoiesis, Iron metabolism, Polycythemia etiology, Rats, Serum Albumin analysis, gamma-Globulins analysis, Blood Proteins analysis, Polycythemia metabolism
- Abstract
Plasma proteins in the period of post-transfusion polycythaemia in rats. Acta Physiol. Pol., 1978, 29 (1): 47-54. The plasma protein of polycythaemic rats with strongly inhibited erythropoiesis and of normal rats was characterized with the use of ion exchanger chromatography on DEAE-cellulose in an NaCl concentration gradient and electrophoresis on polyacrylamide gel. Experimental posttransfusion polycythaemia was found to cause considerable changes in the amount and composition of plasma proteins. The amount of gamma and alpha1 globulins increased in polycythaemic animals, whereas albumin content decreased. There also appeared wide differences in the elution profiles obtained from ion exchanger chromatography of both kinds of plasma. Electrophoretic analysis of fractions obtained by chromatography demonstrated a similarity only in the composition of the globulin fractions of both the plasma kinds. The remaining fractions differed widely in their protein composition.
- Published
- 1978
29. Changes in erythropoietic activity of the rat plasma in the course of posttransfusion polycythaemia.
- Author
-
Goralska M
- Subjects
- Animals, Blood Proteins analysis, Bone Marrow metabolism, Hematocrit, Hemoglobins analysis, Iron metabolism, Male, Polycythemia etiology, Rats, Erythropoiesis, Polycythemia blood, Transfusion Reaction
- Abstract
The aim of this study was to determine the erythropoietic activity of the plasma of polycythaemic rats and of one of its protein fractions playing a role in erythropoiesis regulation. The erythropoietin activity and that of the erythropoiesis inhibitor varied in the examined plasma samples at definite time periods after induction of polycythaemia. It was demonstrated that the most suitable time of plasma collection for the inhibitor investigation is the period between 115 and 187 h after the first transfusion, and that in some cases separation of this factor from erythropoietin present simultaneously in the plasma is indispensable in order to reveal the inhibitory activity. The erythropoiesis inhibitor administered jointly with erythropoietin was found to exert no influence on erythropoiesis either in normal or in polycythaemic recipients of the tested plasma.
- Published
- 1977
30. THe dynamics of erythropoiesis inhibition in posttransfusion polycythaemia in the rat.
- Author
-
Goralska M
- Subjects
- Animals, Bone Marrow metabolism, Iron metabolism, Male, Polycythemia etiology, Rats, Erythropoiesis, Polycythemia physiopathology, Transfusion Reaction
- Abstract
The dynamics of erythropoiesis inhibition as the consequence of polycythaemia induced in rats by four to six transfusions of homologous erythrocytes was studied in detail. It was found, that, in rats with polycythaemia elicited by two transfusions of erythrocytes, erythropoiesis inhibition occurs 67h after the first transfusion and it is most pronounced in the period between 115-187 h. The hemopoietic system is not completely free from the inhibitory influence of polycythaemia up to 283 h after the first transfusion. Maintenance of a state of polycythaemia for 17 days by repeated transfusions strongly and durably inhibits the haemopoietic system. The red blood cell system remained for this whole period under the inhibitory effect.
- Published
- 1977
31. Isolation and purification of porcine LH for radioimmunoassay and radioreceptor assay.
- Author
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Ziecik A, Goralska M, Krzymowski T, and Pogorzelski K
- Subjects
- Animals, Pituitary Gland analysis, Radioimmunoassay, Radioligand Assay, Swine, Luteinizing Hormone isolation & purification
- Published
- 1979
32. The lymphatic system and erythropoiesis. II. Influence of antilymphocytic serum on the process of erythropoiesis. Inhibition in experimental polycythaemia.
- Author
-
Przala J, Krzymowski T, and Goralska M
- Subjects
- Animals, Blood Transfusion, Bone Marrow metabolism, Bone Marrow Cells, Erythrocyte Count, Erythrocytes metabolism, Hematocrit, Hemoglobins, Iron Radioisotopes, Liver metabolism, Lymph Nodes cytology, Lymphocytes immunology, Rabbits, Rats, Rats, Inbred Strains, Schilling Test, Spleen cytology, Spleen metabolism, Antilymphocyte Serum pharmacology, Erythropoiesis, Polycythemia blood
- Published
- 1973
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