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  • 1
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The 17-kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication-defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor-κB (NF-κB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double-stranded oligonucleotide probe containing the consensus binding sequence for NF-κB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF-κB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen-activated protein kinase inhibitor SB-203580 also suppressed the GMF-activated NF-κB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate activated NF-κB whether or not GMF was overexpressed. Along with NF-κB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF-κB nuclear translocation was blocked by its specific decoy DNA, implicating NF-κB as an upstream mediator of this anti-oxidant enzyme. The p38 inhibitor SB-203580 also blocked the GMF-activated SOD. As NF-κB and SOD are both pro-survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor function and eventual death as a result of degeneration of motor neurons in the spinal cord and brain. The discovery of mutations in SOD1, the gene encoding the antioxidant enzyme Cu/Zn-superoxide dismutase (CuZnSOD), in a subset of ALS patients has led to new insight into the pathophysiology of ALS. Utilizing a novel adenovirus gene delivery system, our laboratory has developed a human cell culture model using chemically differentiated neuroblastoma cells to investigate how mutations in SOD1 lead to neuronal death. Expression of mutant SOD1 (G37R) resulted in a time and dose-related death of differentiated neuroblastoma cells. This cell death was inhibited by overexpression of the antioxidant enzyme manganese superoxide dismutase (MnSOD). These observations support the hypothesis that mutant SOD1- associated neuronal death is associated with alterations in oxidative stress, and since MnSOD is a mitochondrial enzyme, suggest that mitochondria play a key role in disease pathogenesis. Our findings in this model of inhibition of mutant SOD1-associated death by MnSOD represent an unique approach to explore the underlying mechanisms of mutant SOD1 cytotoxicity and can be used to identify potential therapeutic agents for further testing.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients. In an in vitro cell culture system, we demonstrated that infection of mouse NSC-34 motor neuron-like cells with adenovirus containing mutant G93A-SOD1 gene increased cellular oxidative stress, mitochondrial dysfunction, cytochrome c release and motor neuron cell death. Cells pretreated with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and synergistically exacerbated motor neuron-like cell death with mutant G93A-SOD1 but not with wild-type SOD1. Similarly, overexpression of mitochondrial antioxidative genes, MnSOD and GPX4 by stable transfection significantly increased NSC-34 motor neuron-like cell resistance to mutant SOD1. Pre-incubation of cells with␣spin trapping molecule, 5′,5′-dimethylpryrroline-N-oxide (DMPO), prevented mutant SOD1-mediated mitochondrial dysfunction and cell death. Furthermore, treatment of mutant G93A-SOD1 transgenic mice with DMPO significantly delayed paralysis and increased survival. These findings suggest a causal relationship between enhanced oxidative stress and mutant SOD1-mediated motor neuron degeneration, considering that enhanced oxygen free radical production results from the SOD1 structural alterations. Molecular approaches aimed at increasing mitochondrial antioxidative activity or effectively blocking oxidative stress propagation can be potentially useful in the clinical management of human ALS disease.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1420-908X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-6903
    Keywords: Glia ; glioma ; maturation ; oxidative stress ; antioxidant enzymes ; superoxide dismutase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to evaluate the intracellular function of glia maturation factor (GMF), we overexpressed GMF in C6 rat glioma cells using two methods: stable transfection using the pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation as shown by the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls, and express the mature astrocytic marker glial fibrillary acidic protein. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent superoxide dismutase (CuZnSOD) activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an induction of the enzyme. In view of recent findings that reactive oxygen species (ROS) and the antioxidant enzymes are intricately involved in key physiologic processes such as proliferation, differentiation and apoptosis, the study raises the possibility that CuZnSOD may be a mediator of GMF function.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Molecular and cellular biochemistry 84 (1988), S. 147-153 
    ISSN: 1573-4919
    Keywords: superoxide ; dismutase ; catalase ; glutathione ; peroxidase ; immortality ; cancer ; mitochondria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Summary The role of antioxidant enzymes, particularly superoxide dismutase (SOD), in immortalization and malignant transformation is discussed. SOD (generally MnSOD) has been found to be lowered in a wide variety of tumor types when compared to an appropriate normal cell control. Levels of immunoreactive MnSOD protein and mRNA for MnSOD also appear to be lowered in tumor cells. Tumor cells have the capacity to produce superoxide radical, the substrate for SOD. This suggests that superoxide production coupled with diminished amounts of MnSOD may be a general characteristic of tumor cells. The levels of MnSOD in certain cells correlates with their degree of differentiation; non-differentiating cells, whether normal or malignant, appear to have lost the ability to undergo MnSOD induction. These observations are used to elucidate a two-step model of cancer. This model involves not only the antioxidant enzymes, but also organelle (particularly mitochondria and peroxisomes) function as a dominant theme in carcinogenesis.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Age 21 (1998), S. 95-97 
    ISSN: 1574-4647
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular histology 26 (1994), S. 734-753 
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Immunoperoxidase and immunogold techniques were used to localize the following antioxidant enzyme systems in the adult hamster kidney at the light and ultrastructural levels: superoxide dismutases, catalases, peroxidases and glutathione S-transferases. Each cell type in the kidney showed specific patterns of labelling of these enzymes. For example, proximal and distal tubular and transitional epithelial cells showed significant staining for all of these enzymes, while glomerular cells and cells of the thin loop of Henle did not show significant staining at the light microscope level. In addition, high levels of glutathione peroxidase were found in smooth muscle cells of renal arteries. At the ultrastructural level, each enzyme was found in a specific subcellular location. Manganese superoxide dismutase was found in mitochondria, catalase was localized in peroxisomes, while copper, zinc superoxide dismutase and glutathione S-transferase (liver and placental forms) were found in both the nucleus and cytoplasm. Glutathione peroxidase was found to have a broad intracellular distribution, with localization in mitochondria, peroxisomes, nucleus, and cytoplasm. Microvilli of tubular cells were labelled by antibodies to catalase, copper, zinc superoxide dismutase, glutathione peroxidase, and glutathione S-transferases. Cell types that were negative by light microscopy immunoperoxidase studies showed definite labelling with immunogold post-embedding ultrastructural techniques (glomerular cells and cells of the loop of Henle), demonstrating the greater sensitivity of the latter technique. These observations demonstrate that there are large variations in the levels of antioxidant enzymes in different cell types, and that even within a distinct cell type, the levels of these enzymes vary in different subcellular locations. Our results demonstrate for the first time the overall antioxidant enzyme status of individual kidney cell types, thereby explaining why different cell types have differing susceptibilities to oxidant stress. Possible physiological and pathological consequences of these findings are discussed.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular histology 27 (1995), S. 575-586 
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Immunolocalization studies of hamster kidney development were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase and glutathioneS-transferases and their subunits. Antibodies to extracellular matrix proteins were also studied to determine the temporal sequence between expression of immunoreactive protein for basement membrane proteins, which serve as markers of embryonic induction of nephron development, and antioxidant enzyme expression in kidney development. Immunoreactive proteins for antioxidant enzymes were not detectable in the developing kidney until after extracellular matrix proteins had been deposited. However, immunoreactive proteins for the antioxidant enzymes copper, zinc and manganese superoxide dismutases, catalase, and α class glutathioneS-transferase Ya subunit were detected in renal tubules before birth. μ class glutathioneS-transferase subunits Yb1 and Yb2 stained transitional epithelium at high levels before birth. Our results indicate: (1) each type of kidney cell has a unique antioxidant enzyme profile, (2) antioxidant enzymes are expressed in different types of cell at different times during development, but antioxidant enzyme immunoreactive protein was not present until after immunoreactive proteins for extracellular matrix molecules were detected, and (3) certain antioxidant enzymes are present before birth, indicating that high oxygen tension present at birth is not crucial for induction of immunoreactive protein.
    Type of Medium: Electronic Resource
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