Han Geuk Seo

Inactivation of Glutathione Peroxidase by Nitric Oxide IMPLICATION FOR CYTOTOXICITY

S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC of SNAP for GPx was 2 μM at 1 h of incubation and was 20% of the IC for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly inactivated the enzyme. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 μM SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N-methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO. This mechanism may at least in part explain the cytotoxic effects of NO on cells and NO-induced apoptotic cell death.

Characterization of Wild‐Type and Amyotrophic Lateral Sclerosis‐Related Mutant Cu,Zn‐Superoxide Dismutases Overproduced in Baculovirus‐Infected Insect Cells

Abstract: We describe the use of a baculovirus expression system to overproduce human Cu,Zn‐superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn‐SOD cDNA synthesized a large amount of Cu,Zn‐SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose‐dependent manner when Cu2+ and Zn2+ were added to the medium. Cells grown in media supplemented with Cu2+ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu2+ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn‐SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti‐human Cu,Zn‐SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly41Asp, His43Arg, and Gly85Arg exhibited 47, 66, and 99% of wild‐type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.

Roles of Purine Nucleotides and Adenosine in Enhancing NOS II Gene Expression in Interleukin-1β-Stimulated Rat Vascular Smooth Muscle Cells

The production of nitric oxide (NO) by vascular smooth muscle cells (VSMC) is stimulated by interleukin-1 beta (IL-1 beta). This is enhanced in a dose-dependent manner by ADP, although it alone failed to induce nitrite accumulation. Purine nucleotides and their nonhydrolizable analogues as well as adenosine also exhibit variable enhancing effects. This enhanced nitrite formation was due to induction of the NO synthase (NOS II) gene as judged by Northern hybridization using an NOS II specific probe and by Ca2+ independency of the NOS II activity. 8-(p-Sulfophenyl)-theophylline, a blocker of adenosine receptors, suppressed the enhanced NO production by adenosine and ADP to the level of that with IL-1 beta alone. These data indicate that activation of the adenosine receptor on VSMC may enhance production of NOS II by modulating a signal transducing pathway of IL-1 beta. Although cAMP is a candidate as the second messenger, it was not significantly elevated by either ADP or adenosine treatment in IL-1 beta-stimulated cells. This mechanism might be stimulated under conditions with release of various purine and their derivatives.

Differential Expression of Mn- and Cu,Zn-Superoxide Dismutases in Various Tissues of LEC Rats

Oxy-radicals are thought to play important roles in the course of multistep carcinogenesis [1–3]. Accurate detection of oxy-radicals is impossible, however, because of their instability and short half-life. Therefore, evaluation of the levels of enzymes involved in the metabolism of oxy-radicals appears to be the most reliable means of assessing oxy-radical metabolism in vivo.

Mutual interaction between oxidative stress and endoplasmic reticulum stress in the pathogenesis of diseases specifically focusing on non-alcoholic fatty liver disease

Reactive oxygen species (ROS) are produced during normal physiologic processes with the consumption of oxygen. While ROS play signaling roles, when they are produced in excess beyond normal antioxidative capacity this can cause pathogenic damage to cells. The majority of such oxidation occurs in polyunsaturated fatty acids and sulfhydryl group in proteins, resulting in lipid peroxidation and protein misfolding, respectively. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) is enhanced under conditions of oxidative stress and results in ER stress, which, together, leads to the malfunction of cellular homeostasis. Multiple types of defensive machinery are activated in unfolded protein response under ER stress to resolve this unfavorable situation. ER stress triggers the malfunction of protein secretion and is associated with a variety of pathogenic conditions including defective insulin secretion from pancreatic β-cells and accelerated lipid droplet formation in hepatocytes. Herein we use nonalcoholic fatty liver disease (NAFLD) as an illustration of such pathological liver conditions that result from ER stress in association with oxidative stress. Protecting the ER by eliminating excessive ROS via the administration of antioxidants or by enhancing lipid-metabolizing capacity via the activation of peroxisome proliferator-activated receptors represent promising therapeutics for NAFLD.

The viability of primary hepatocytes is maintained under a low cysteine-glutathione redox state with a marked elevation in ophthalmic acid production

Abstract Extracellular cystine, the oxidized form of cysteine (Cys), is taken up by cells via the cystine transporter xCT. xCT is not expressed in the liver but is induced in primary hepatocytes under conventional cultured conditions. However, compared to wild‐type hepatocytes those from the xCT‐knockout mouse showed no evidence of an abnormality and the levels of both Cys and glutathione (GSH) remained unchanged. The levels of ophthalmic acid (OPT), which is produced as an alternative compound by the GSH‐synthesizing pathway, became increased during the culturing of hepatocytes. It therefore appears that, in primary hepatocytes, Cys is provided by systems other than xCT, most likely via the transsulfuration pathway, but the levels that are produced are not sufficient. We also employed mouse hepatoma‐derived Hepa1‐6 cells, which constitutively express xCT. When Hepa 1‐6 cells were cultivated in Cys‐free media, the levels of intracellular Cys and GSH were decreased, compared to cells cultured in conventional media, leading to cell death accompanied by an increase in the levels of reactive oxygen species and lipid peroxidation products with characteristics similar to ferroptosis. While OPT levels were increased by only to a limited extent in Hepa 1‐6 cells, primary hepatocytes cultured in Cys‐ and Met‐free media showed a marked elevation in OPT, reaching levels nearly equivalent to the GSH levels when the cells were cultured in conventional media. Thus, OPT may become a marker for Cys insufficiency and might be used to predict pathological conditions of cells with elevated oxidative stress. Graphical abstract Differential cysteine‐supply systems between the primary mouse hepatocytes and Hepa 1‐6 cells under conventional or Cys/Met‐free culture. Met; Methionine, Hcy; Homocysteine, Cst; Cystathionine, Cys; Cysteine, Thr; Threonine, Glu; Glutamate, 2OB; 2‐Oxo‐butyric acid, 2AB; 2‐Amino‐butyric acid, GSH; Glutathione, OPT; Ophthalmic acid, &ggr;GCS; &ggr;‐Glutamylcysteine synthetase, GS; Glutathione synthetase; NAAT; neutral amino acid transporter Figure. No Caption available. HighlightsxCT is induced in primary mouse hepatocytes under culture conditions.xCT‐KO hepatocytes contained normal levels of cysteine and glutathione (GSH).Primary hepatocytes were viable in cysteine‐ and methionine‐free media.Ophthalmic acid levels were equivalent to those of GSH in hepatocytes.