Nobuo Ueta

Lethal forebrain ischemia stimulates sphingomyelin hydrolysis and ceramide generation in the gerbil hippocampus.

Ceramide, a hydrolyzed product of sphingomyelin, is reported to play an important role in apoptosis. In this study, we measured the sphingomyelin and ceramide levels in the hippocampus of the gerbil after transient forebrain ischemia for 5 min (lethal) or 2 min (sublethal). The aim was to examine alterations in the sphingomyelin cycle during delayed neuronal death, which we considered could be due to apoptosis. Sphingolipids were separated on high-performance thin-layer chromatography plates and analyzed by gas-liquid chromatography. At 30 min and 24 h after lethal ischemia, sphingomyelin levels were decreased and ceramide levels were increased compared with control levels. No significant changes were observed after sublethal ischemia. These results suggest that the sphingomyelin cycle may have a role in neuronal death.

Fatty acid composition of the chronic subdural hematoma: with reference to its recurrence

The fatty acid composition of aspirated chronic subdural hematoma (CSDH) was measured by gas liquid chromatography and the relationship between fatty acid and recurrence of the hematoma was assessed. Thirty patients with CSDH were operated on through a single burr-hole; 4 patients developed recurrent hematoma (13%). The lipid composition of CSDH was mainly phosphatidylcholine, sphingomyelin, cholesterol, free fatty acid, triacylglycerol and cholesterol ester. The fatty acid constituents were palmitic, stearic, oleic, linoleic, arachidonic and docosahexanoeic acids. Analysis of the polyunsaturated fatty acids demonstrated that hematoma taken from patients with recurrent CSDH contained more linoleic acid (n-6) than those with non-recurrent CSDH. Linoleic and arachidonic acids are known to induce angiogenesis in cultured aortic endothelial cells. Change in fatty acid composition of recurrent hematoma might be associated with rebleeding from the hematoma capsule.

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation.

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.

Oxidized low-density lipoprotein induces the production of superoxide by neutrophils

Exposure of guinea pig peritoneal neutrophils to ox‐LDL led to the production of superoxide, which was measured by the formation of superoxide‐dependent chemiluminescence. The cells exposed to unoxidized LDL, e.g. native LDL, acetyl‐LDL, and self‐aggregates of LDL showed no production of superoxide. The superoxide production was correlated with the levels of oxidative modification of LDL and reached a maximum between 10 and 30 min during incubation, but preincubating the cells with cytochalasin B decreased the superoxide production. These findings indicate that neutrophils rapidly take up ox‐LDL by phagocytosis and generate superoxide which may cause superoxide‐mediated lipid peroxidation in vivo.

Antioxidative Activity of 3,4-Dihydroxyphenylacetic Acid and Caffeic Acid in Rat Plasma

The purpose of the present paper is to study and compare in vitro the inhibitory effect of 3,4-dihydroxyphenylacetic acid (DOPAC) and caffeic acid (CA) on lipid peroxidation in rat plasma. Rat plasma was oxidized at 37°C by the radical initiators 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) or 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). The consumption of endogenous α-tocopherol (α-TOH) and the accumulation of conjugated diene hydroperoxides were measured by high-performance liquid chromatography and by ultraviolet spectroscopy, respectively. α-TOH was consumed at the same rate in the presence of 20 mM AAPH or 2 mM MeO-AMVN. DOPAC and CA suppressed the α-TOH consumption in a dose-dependent manner. A concentration of 50 μM of both phenolic acids was sufficient to induce a lag phase and to delay the rate of α-TOH consumption. The effect was more pronounced in rat plasma oxidation by AAPH than by MeO-AMVN. CA spared vitamin E more effectively than DOPAC in both oxidations. DOPAC and CA suppressed the formation of conjugated diene hydroperoxides. DOPAC and CA at concentration 50 μM suppressed α-TOH consumption during oxidation of soybean phosphatidylcholine (2.8 mM) multilamellar vesicles containing 15 μM α-TOH, in which the lipophilic initiator 2,2′-azobis (2,4-dimethylvaleronitrile) (6 mM) was incorporated. In conclusion, we demonstrated that DOPAC and CA in micromolar concentrations have antioxidant activity in rat plasma, a medium very close to the conditions in vivo, suggesting that supplementation with the phenolic acids will provide significant antioxidant protection.

Isolation and analysis of age-related fluorescent substances in rat testes.

Fluorescent substances were extracted from rat testicular tissue with 2,2-dimethoxypropane (DMP) and analyzed by 2-dimensional thin layer chromatography (TLC). One substance that accumulated with increasing age of the animals was isolated and analyzed quantitatively by spectrophotofluorometry using quinine sulfate as a standard. This substance, which was designated as an age-related fluorescent substance (ARFS), exhibited an excitation maximum at 355 nm and an emission maximum at 490 nm. Its fluorescence was quenched by metal chelators and at alkaline pH, indicating it contained a conjugated Schiff base structure. Quantitative analysis of this substance in the testes of rats 1, 2, 11 and 20 months of age showed that it increased linearly with age. The relation of this substance to aging also was indicated by its detection in animals of different ages fed diets of both low and high unsaturation.

Regulatory functions of cyclic adenosine 3',5'-monophosphate in 1-methyladenine production by starfish follicle cells

The biosynthesis of 1-methyladenine (1-MeAde) in follicle cells of the starfish, Asterina pectinifera, occurred in response to a gonad-stimulating substance (GSS). Simultaneously with 1-MeAde production, the intracellular cAMP level immediately increased following the administration of GSS. This level in follicle cells markedly depended on GSS concentration. Although 1-MeAde production was also induced by 1-methyladenosine, it caused no increase in cAMP content. It thus appears that the effect of GSS on starfish follicle cells results in the receptor-mediated formation of cAMP.

Conformational changes in oxidized LDL recognized by mouse peritoneal macrophages.

Mouse peritoneal macrophages have been considered to recognize and take up oxidized LDL by a scavenger receptor. However, it is still unknown what conformational changes in oxidized LDL contribute to recognition by the macrophage scavenger receptor. In the present study, it was shown that the amount of oxidized LDL taken up by macrophages correlated well with the fluorescence intensity formed in oxidized LDL. The autofluorescent products generated in oxidized LDL were characterized by Ex:365 nm Em:430 nm, and the intensity of the fluorescence was reduced at base pH, and restored by adjusting the pH to neutral. The characteristics of the fluorescent products indicate that a Schiff base structure was formed in oxidized LDL. Oxidized LDLs were fractionated into native size and aggregated large particles with HPLC by monitoring fluorescence. It was demonstrated that macrophages ingest selectively or preferentially aggregated oxidized LDL, but not native size oxidized LDL. The incorporation of aggregated oxidized LDL was remarkably suppressed by heparin and cytochalasin B. These results suggest that mouse peritoneal macrophages recognize the conformational changes in oxidized LDL related to the formation of a Schiff base structure with increasing autofluorescence, and ingest selectively aggregated large particles in oxidized LDL in a phagocytic process.

Age and organ difference in amount and distribution of autofluorescent granules in rats

The amount and distribution of autofluorescent granules in various organs and tissues of rats of different ages (2-, 11-, and 29.5-month-old) were compared by morphometrical analysis. The age-related increase of the granules was observed in the cardiac muscle and hepatic cells, but the amount of granules was little even in 29.5-month-old rats. In some other organs, the granules appeared early, at 2 months of age, increased up to 11 months, but thereafter no significant increase was observed. The autofluorescent granules are not merely considered to be an age-related pigment, but seems to be influenced by relationship to the cell metabolism and other functions.

Ethanolamine plasmalogen and cholesterol reduce the total membrane oxidizability measured by the oxygen uptake method.

To investigate the effects of ethanolamine plasmalogen, phosphatidylethanolamine, cholesterol, and alpha-tocopherol on the oxidizability of membranes, various large unilamellar vesicles (LUVs) including these lipids and antioxidant were examined for their total membrane oxidizabilities, evaluated as R(p)/R(i)(1/2) value (where R(p) is rate of oxygen consumption and R(i)(1/2) is the square root of rate of chain initiation) by the oxygen uptake method with water-soluble radical initiator and inhibitor. Incorporation of bovine brain ethanolamine plasmalogen (BBEP) into vesicles as well as cholesterol led to lower the total membrane oxidizability dose-dependently. The effect of BBEP was more efficient in the presence of cholesterol in vesicles. On the other hand, diacyl counterpart, egg yolk phosphatidylethanolamine, and a typical radical scavenger, alpha-tocopherol, had no effect on the membrane oxidizability. Alpha-tocopherol only prolonged an induction period dose-dependently in the present oxidizing system, suggesting a novel antioxidant mechanism of ethanolamine plasmalogens besides the action of scavenging radicals.