Dai-Eun Sok

Oxidative Inactivation of Paraoxonase1, an Antioxidant Protein and its Effect on Antioxidant Action

Paraoxonase1 (PON1), one of antioxidant proteins to protect low density lipoprotein (LDL) from the oxidation, is known to lose its activity in the oxidative environment. Here, we attempted to elucidate the possible mechanisms for the oxidative inactivation of PON1, and to examine the capability of hydroxyl radicals-inactivated PON1 to prevent against LDL oxidation. Of various oxidative systems, the ascorbate/Cu2+ system was the most potent in inactivating the purified PON1 (PON1) as well as HDL-bound PON1 (HDL-PON1). In contrast to a limited inactivation by Fe2+ (2.0 μM), the inclusion of Cu2+ (0.1–1.0 μM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.5 mM). A similar result was also obtained with the inactivation of HDL-PON1. The inactivation of PON1 by ascorbate/Cu2+ was pevented by catalase, but not general hydroxyl radical scavengers, supporting Cu2+-catalyzed oxidative inactivation. In addition, Cu2+ alone inactivated PON1, either soluble or HDL-bound, by different mechanisms, concentration-dependent. Separately, there was a reverse relationship between the inactivation of PON1 and its preventive action against LDL oxidation during Cu2+-induced oxidation of LDL. Noteworthy, ascorbate/Cu2+-inactivated PON1, which was charaterized by the partial loss of histidine residues, expressed a lower protection against Cu2+-induced LDL oxidation, compared to native PON1. Based on these results, it is proposed that metal-catalyzed oxidation may be a primary factor to cause the decrease of HDL-associated PON1 activity under oxidative stress, and radicals-induced inactivation of PON1 may lead to the decrease in its antioxidant action against LDL oxidation.

Beneficial effect of oleoylated lipids on paraoxonase 1: protection against oxidative inactivation and stabilization.

The effect of lipids on PON1 (paraoxonase 1), one of antioxidant proteins in high-density lipoprotein, was investigated in respect to inhibition, protection against oxidative inactivation, and stabilization. When the effect of lipids on the PON1 activity was examined, a remarkable inhibition was expressed by polyenoic fatty acids (C18:2-C20:5). Linoleic acid, the most potent ( K(i), 3.8 microM), showed competitive inhibition. Next, various lipids were examined for prevention against the inactivation of PON1 by ascorbate/Cu2+, which caused a remarkable (>or =90%) inactivation of PON1. Compared with saturated fatty acids (C6-C18), exhibiting a modest protection (9-40%), monoenoic acids (C16:1-C20:1) showed a greater maximal protective effect (Emax, 70-82%), with oleic acid being the most effective (EC50, 2.7 microM). In contrast, polyenoic acids showed no protection. Noteworthy, linoleic acid prohibited the protective action of oleic acid non-competitively. In the structure-activity relationship, a negatively charged group seems to be required for the protective action. Consistent with this, dioleoylphosphatidylglycerol, negatively charged, was more protective than dioleoylphosphatidylcholine. These data, together with requirement of Ca2+ (EC50, 0.6 microM) for the protective action, may support the existence of a specific site responsible for the protective action. A similar protective action of lipids was also observed in the inactivation of PON1 by ascorbate/Fe2+, peroxides or p -hydroxymercuribenzoate. Separately, PON1 was stabilized by oleic acid or oleoylated phospholipids, in combination with Ca2+, but not linoleic acid. These results suggest that in contrast to an adverse action of linoleic acid, monoenoic acids or their phospholipid derivatives play a beneficial role in protecting PON1 from oxidative inactivation as well as in stabilizing PON1.

Organophosphate-induced brain injuries: delayed apoptosis mediated by nitric oxide

The features of organophosphate-induced brain injuries were investigated. Rats were poisoned intraperitoneally with 9 mg/kg (1.8 LD(50)) of diisopropylfluorophosphate. Pyridostigmine bromide (0.1 mg/kg) and atropine methylnitrate (20 mg/kg), which are centrally inactive, were pre-treated intramuscularly to reduce the mortality and eliminate peripheral signs. Diisopropylfluorophosphate induced severe limbic seizures, and early necrotic and delayed apoptotic brain injuries. The necrotic brain injury was observed to be maximal as early as 1 h after diisopropylfluorophosphate treatment predominently in hippocampus and piriform/entorhinal cortices, showing a spongiform change (malacia) of neuropils in severe cases. In contrast, typical apoptotic (TUNEL-positive) cells started to appear at 12 h in thalamus, and a mixed type in amygdala. Separately, nitrite/nitrate content in cerebrospinal fluid was found to significantly increase after 2 h, reaching a maximal level at 6 h. Pre-treatment with l-N(G)-nitroarginine, an inhibitor of nitric oxide synthase, reduced nitrite/nitrate content and, noteworthy, attenuated only apoptotic brain injury in all four brain regions without affecting seizure intensity and necrotic injury. Taken together, the delayed apoptotic injury of brain induced by diisopropylfluorophosphate poisoning in rats might be mediated in part through nitric oxide production.

Ethanol fermentation from Jerusalem artichoke powder using Saccharomyces cerevisiae KCCM50549 without pretreatment for inulin hydrolysis

A strain of Saccharomyces cerevisiae, KCCM50549, was found to efficiently ferment the inulin-containing carbohydrates in Jerusalem artichoke without acidic or enzymatic pretreatment prior to fermentation. S. cerevisiae KCCM50549 could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke (up to degree of polymerization (DP) of 15), in contrast to the other S. cerevisiae strain such as NCYC625 that fermented the fructo-oligosaccharides with DP of up to around six. Inulin-fermenting S. cerevisiae KCCM50549 produced c.a. 1.6 times more ethanol from Jerusalem artichoke compared with S. cerevisiae NCYC625. Direct ethanol fermentation of Jerusalem artichoke flour at 180 g/L without any supplements or pretreatments by S. cerevisiae KCCM50549 in a 5 L jar fermentor yielded 36.2 g/L of ethanol within 36 h. The conversion efficiency of inulin-type sugars to ethanol was 70% of the theoretical ethanol yield.

In Vitro Antioxidant and Anti-inflammatory Activities of Jaceosidin from Artemisia princeps Pampanini cv. Sajabal

Oxidized low-density lipoprotein (oxLDL) plays a key role in the inflammatory processes of atherosclerosis. Jaceosidin isolated from the methanolic extracts of the aerial parts of Artemisia princeps Pampanini cv. Sajabal was tested for antioxidant and anti-inflammatory activities. Jaceosidin inhibited the Cu2+-mediated LDL oxidation with IC50 values of 10.2 μM in the thiobarbituric acid-reactive substances (TBARS) assay as well as the macrophage-mediated LDL oxidation. The antioxidant activities of jaceosidin were exhibited in the conjugated diene production, relative electrophoretic mobility, and apoB-100 fragmentation on copper-mediated LDL oxidation. Jaceosidin also inhibited the generation of reactive oxygen species (ROS) concerning in regulation of NF-κB signaling. And jaceosidin inhibited nuclear factor-kappa B (NF-κB) activity, nitric oxide (NO) production, and suppressed expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.

Hepatoprotective effect of aged black garlic on chronic alcohol-induced liver injury in rats.

The hepatoprotective effect of aged black garlic (ABG) against ethanol-induced oxidative liver damage was investigated in adult male Sprague-Dawley rats for 4 weeks. Rats were divided into three groups: a saline (WT) group, an ethanol (ET) group (15 mL/kg of body weight 20% [wt/vol] ethanol), and an ethanol + ABG (ET+ABG) group (ethanol + 100 mg/kg of body weight ABG). ABG administration led to decreased epididymal and total fat pad (P<.05) and liver weights, ameliorated prominent fatty changes around the portal triad, and reduced fat accumulation in liver. ABG caused a significant decrease of the alcohol-induced increases in hepatic activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase. Cytochrome P450 2E1 activity was reduced by 55%, whereas the activities of glutathione S-transferase and quinine reductase were increased by 1.5-fold (P<.05) and fourfold (P<.05), respectively, in the ET+ABG group compared with the ET group. ABG treatment significantly decreased the thiobarbituric acid-reactive substances level in liver, heart, and plasma. Glutathione content and the activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, and catalase in liver were significantly enhanced. Furthermore, the oxidative damage to blood lymphocyte DNA caused by chronic alcohol ingestion was significantly decreased in the ET+ABG group. In conclusion, ABG has strong antioxidative properties and may be a promising agent for protecting against chronic alcohol-induced liver damage.

Oxygenation of arachidonoyl lysophospholipids by lipoxygenases from soybean, porcine leukocyte, or rabbit reticulocyte.

Oxygenation of arachidonoyl lysophosphatidylcholine (lysoPC) or arachidonoyl lysophosphatidic acid (lysoPA) by lipoxygenase (LOX) was examined. The oxidized products were identified by HPLC/UV spectrophotometry/mass spectrometry analyses. Straight-phase and chiral-phase HPLC analyses indicated that soybean LOX-1 and rabbit reticulocyte LOX oxygenated arachidonoyl lysophospholipids mainly at C-15 with the S form as major enantiomer, whereas porcine leukocyte LOX oxygenated at C-12 with the S form. Next, the sequential exposure of arachidonoyl-lysoPC to soybean LOX-1 and porcine leukocyte LOX afforded two major isomers of dihydroxy derivatives with conjugated triene structure, suggesting that 15(S)-hydroperoxyeicosatetraenoyl derivatives were converted to 8,15(S)-dihydroxyeicosatetraenoyl derivatives. Separately, arachidonoyl-lysoPA, but not arachidonoyl-lysoPC, was found to be susceptible to double oxygenation by soybean LOX-1 to generate a dihydroperoxyeicosatetraenoyl derivative. Overall, arachidonoyl lysophospholipids were more efficient than arachidonic acid as LOX substrate. Moreover, the catalytic efficiency of arachidonoyl-lysoPC as substrate of three lipoxygenases was much greater than that of arachidonoyl-lysoPA or arachidonic acid. Taken together, it is proposed that arachidonoyl-lysoPC or arachidonoyl-lysoPA is efficiently oxygenated by plant or animal lipoxygenases, C12- or C15-specific, to generate oxidized products with conjugated diene or triene structure.

Mechanisms for anti-inflammatory effects of 1-[15(S)-hydroxyeicosapentaenoyl] lysophosphatidylcholine, administered intraperitoneally, in zymosan A-induced peritonitis

BACKGROUND AND PURPOSE Lysophosphatidylcholines (lysoPCs) with polyunsaturated acyl chains are known to exert anti‐inflammatory actions. 15‐Lipoxygeanation is crucial for anti‐inflammatory action of polyunsaturated acylated lysoPCs. Here, the anti‐inflammatory actions of 1‐(15‐hydroxyeicosapentaenoyl)‐lysoPC (15‐HEPE‐lysoPC) and its derivatives were examined in a mechanistic analysis.

Spirulina improves antioxidant status by reducing oxidative stress in rabbits fed a high-cholesterol diet.

The beneficial effect of Spirulina (Spirulina platensis) on tissue lipid peroxidation and oxidative DNA damage was tested in the hypercholesterolemic New Zealand White rabbit model. After hypercholesterolemia was induced by feeding a high cholesterol (0.5%) diet (HCD) for 4 weeks, then HCD supplemented with 1% or 5% Spirulina (SP1 or SP5, respectively) was provided for an additional 8 weeks. Spirulina supplementation significantly reduced the increased lipid peroxidation level in HCD-fed rabbits, and levels recovered to control values. Oxidative stress biomarkers such as glutathione, glutathione peroxidase, glutathione reductase, and glutathione S-transferase were significantly improved in the liver and red blood cells of rabbits fed SP1. Furthermore, SP5 induced antioxidant enzyme activity by 3.1-fold for glutathione, 2.5-fold for glutathione peroxidase, 2.7-fold for glutathione reductase, and 2.3-fold for glutathione S-transferase in liver, compared to the HCD group. DNA damage in lymphocytes was significantly reduced in both the SP1 and SP5 groups, based on the comet assay. Findings from the present study suggest that dietary supplementation with Spirulina may be useful to protect the cells from lipid peroxidation and oxidative DNA damage.

Effect of endocannabinoids on IgE-mediated allergic response in RBL-2H3 cells.

Recently, some endocannabinoids were reported to show anti-inflammatory and anti-allergic activities. In this respect, various arachidonoyl endocannabinoids were screened for the inhibition of allergic response in IgE-activated RBL-2H3 cells. Among arachidonoyl endocannabinoids with a low cytotoxicity, only NA-5HT remarkably inhibited the release of β-hexosaminidase (IC(50), 13.58 μM), a marker of degranulation, and tumor necrosis factor-α (IC(50), 12.52 μM), a pro-inflammatory cytokine, in IgE-activated RBL-2H3 cells. Additionally, NA-5HT markedly suppressed the formation of prostaglandin D(2) (PGD(2)) with IC(50) value of 1.27 μM and leukotriene B(4) (LTB(4)) with IC(50) value of 1.20 μM, and slightly LTC4. When effect of NA-5HT on early stage of FcεRI cascade was investigated, it significantly inhibited phosphorylation of Syk, but not Lyn. Furthermore, NA-5HT suppressed phosphorylation of PLCγ1/2 and PKCδ, related to degranulation process, as well as phosphorylation of LAT, ERK1/2, p38, JNK, Gab2, PI3K and Akt, implicated in the expression of pro-inflammatory cytokines. Relative to its effect on the late stage, NA-5HT slightly reduced phosphorylation of 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2). Additionally, NA-5HT significantly reduced the level of p40(phox), and partially inhibited the expression of p47(phox) and p67(phox). From these results, it is suggested that NA-5HT expresses anti-allergic action by suppressing the activation of Syk, LAT, p38, JNK, PI3K and Akt, as well as the expression of ERK1/2 and NADPH oxidase subunits. Further, a strong inhibition of PGD(2) or LTB(4) biosynthesis by NA-5HT may be an additional mechanism for its anti-allergic action. Such anti-allergic actions of NA-5HT may contribute to further information about its biological functions.