Kazumi Ogata

Effects of age on levels of cysteine, glutathione and related enzyme activities in livers of mice and rats and an attempt to replenish hepatic glutathione level of mouse with cysteine derivatives

There was a large statistically significant decrease in the hepatic level of cysteine and glutathione (GSH) in 24 month-old mice compared to young mice, while, cystine and glutathione disulfide (GSSG) contents in the liver did not differ between young and old mice. Activities of cystathionine gamma-lyase and beta-synthase in mouse liver of the 24 month-old group were significantly decreased. In rats, the hepatic levels of cysteine, cystine, GSH and GSSG exhibited no statistically significant change during aging to 30 month. As the rats matured, total hepatic activities of both cystathionine gamma-lyase and beta-synthase increased with maximum levels at 24 months of age and decreased to the same level found in 5 week old for the former and to 22% of that in 5 week old for the latter. Intraperitoneal administration of diethyl maleate to mice led to depletion of hepatic GSH. When N-acetylcysteine and a thiazolidine derivative were intravenously injected after diethyl maleate administration, the hepatic GSH level of mice was restored to the normal level.

The antioxidant ESeroS-GS inhibits NO production and prevents oxidative stress in astrocytes.

Within the central nervous system uncontrolled production of large amounts of nitric oxide (NO) by activated glial cells might be the common pathogenesis of several neurodegenerative disorders, including Alzheimers disease and Parkinsons disease. In the present investigation, we measured the effect of a novel antioxidant gamma-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinyl-glycine sodium salt (ESeroS-GS) on NO production in cultured rat astrocytes. Upon stimulation with 1 microg/mL lipopolysaccharide plus 100 U/mL interferon-gamma which induced the expression of inducible nitric oxide synthase, cultured astrocytes generated large amounts of NO as measured by nitrite assay and ESR technique. The endogenous NO caused oxidative damage in astrocytes, which was confirmed by the accumulation of both cytosolic and extracellular peroxides, the decrease in the cellular glutathione level, and the formation of thiobarbituric acid reactive substrates. Production of endogenous NO resulted in cell death finally. Pretreatment with the novel antioxidant ESeroS-GS effectively decreased the expression of iNOS gene, inhibited the formation of endogenous NO, and prevented NO-induced oxidative damage and cell death in astrocytes. The results suggest that ESeroS-GS might be used as a potential agent for the prevention and therapy of diseases associated with the overproduction of NO by activated astrocytes.

ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts.

The binding of lipopolysaccharides (LPS) to macrophages results in inflammatory responses. In extreme cases it can lead to endotoxic shock, often resulting in death. A broad range of antioxidants, including tocopherols, can reduce LPS activity in vitro and in vivo. To elucidate the underlying mechanisms of their action, we investigated the effect of the sodium salt of γ-L-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-L-cysteinylglycine (ESeroS-GS), a novel α-tocopherol derivative, on LPS-induced inflammation in vitro and in vivo. ESeroS-GS reduced the transcription of TNF-α, IL-1β, IL-6 and iNOS genes in a dose-dependent manner in RAW264.7 macrophages, and inhibited the release of these inflammatory factors. In addition, ESeroS-GS inhibited LPS-induced mortality in a mouse sepsis model. Electrophoretic mobility shift assays (EMSA) and reporter gene assays revealed that ESeroS-GS down-regulated the transcriptional activity of NF-κB. By analyzing the partitioning of CD14 and Toll-like receptor 4 (TLR-4) in cell membrane microdomains, we found that ESeroS-GS attenuates the binding of LPS to RAW264.7 cells via interfering with the relocation of CD14 and TLR-4 to lipid rafts, blocking the activation of interleukin-1 receptor-associated kinase 1 (IRAK-1), and inhibiting the consequent phosphorylation of TAK1 and IKKα/β, which together account for the suppression of NF-κB activation. Taken together, our data suggest that ESeroS-GS can modulate LPS signaling in macrophages by impairing TLR-4 complex assembly via a lipid raft dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Inhibition of platelet aggregation by S-(1,2-dicarboxyethyl)glutathione, intrinsic tripeptide in liver, heart, and lens

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or bakers yeast showed strong inhibitory effects on blood coagulation and platelet aggregation. The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate. DCE-GS did not show chelating activity. As for ADP- or thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation. DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen. The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol-13-acetate. Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different.

The α-tocopherol derivative ESeroS-GS induces cell death and inhibits cell motility of breast cancer cells through the regulation of energy metabolism

Cancer cells are known to exhibit different hallmarks compared with normal cells. Therefore, targeting these features may improve the response to cancer therapy. In this study, we provided direct evidence that the α-tocopherol derivative ESeroS-GS inhibited the viability, migration, and invasion of breast cancer cells. ESeroS-GS induced cell death in different cancer cells in a dose-dependent manner but showed no significant effects on MCF-10A mammary epithelial cells. Although the ESeroS-GS-induced cell death in MDA-MB-231 breast cancer cells was accompanied with the generation of reactive oxygen species and the down regulation of mitochondrial membrane potential (MMP), no such effect on reactive oxygen species and MMP was seen in MCF-10A cells. Further studies indicated that ESeroS-GS down-regulated the expression of hexokinase II, SDH B, UQCRC2 and COX II in MDA-MB-231 cells but not in MCF-10A cells. The down-regulation of these enzymes accounts for the decreased oxidative phosphorylation (OXPHOS) and glycolysis in MDA-MB-231 cells upon ESeroS-GS treatment. We also found that sub-toxic concentration of ESeroS-GS treatment resulted in the impairment of F-actin cytoskeleton assembly and the consequently decreased migratory and invasive ability of MDA-MB-231 cells, which might be due to the inhibition of cellular energy metabolism. These results indicate that ESeroS-GS shows potential to become a novel anti-cancer agent by targeting the energy metabolism of cancer cells.

Effect of EPC-K1 on Myocardial Infarct Size in a Rat Model with Coronary Artery Occlusion and Reperfusion

1. The ability of EPC-K1 to improve myocardial infarction was evaluated in a rat model with coronary artery occlusion and reperfusion. 2. The myocardial infarct size was 30.5 +/- 1.7% by intravenous (i.v.) administration and 28.8 +/- 2.7% by intraduodenal (i.d.) administration of the saline EPC-K1 at doses between 1 and 10 mg/kg, i.v. and at doses between 50 and 200 mg/kg, i.d., reduced myocardial infarct size dose dependently. Significant reduction in myocardial infarct size was found at a dose of 10 mg/kg, i.v., and 200 mg/kg, i.d.

Ophthalmic and norophthalmic acid in lens, liver, and brain of higher animals

A sensitive high-performance liquid chromatographic method for the determination of nanomole levels of ophthalmic and norophthalmic acid has been described. The procedure is based upon the conversion of amino group to 2,4-dinitrophenyl derivatives and the detection at 420 nm. This method was applied to the determination of the peptides in lens, liver, and brain of several animals. Mean recoveries were 93.6% for ophthalmic acid and 92.3% for norophthalmic acid added to cattle lens homogenate.