Peter P.K. Ho

The antioxidant LY231617 reduces global ischemic neuronal injury in rats.

Background and Purpose In the rat four-vessel occlusion model with 30 minutes of ischemia most agents have failed to be of benefit when given after ischemia. Because postischemia administration is more clinically relevant, we evaluated the antioxidant LY231617 (2,6-bis(1,1-dimethylethyl)-4-[[(1-ethyl)ami-no]methyl]phenol hydrochloride]) when administered after 30 minutes of four-vessel occlusion. Methods Male Wistar rats were subjected to 30 minutes of four-vessel occlusion. LY231617 was either given orally 30 minutes before ischemia or intravenously beginning at 30 minutes after the onset of ischemia. Hippocampal CA1 layer and striatal damage were rated on a scale of 0-3 (0, no damage; 3, >90% cell loss). We also evaluated the ability of LY231617 to prevent iron-dependent lipid peroxidation and to prevent hydrogen peroxide-induced neuronal death of hippocampal neurons in primary culture by exposing cultures to a 50-¨M concentration of hydrogen peroxide for 15 minutes in the presence of LY231617. Results Oral administration of LY231617 reduced both striatal and hippocampal CA1 damage by >75% (p<0.0001). In two separate experiments in which LY231617 was given intravenously beginning 30 minutes after occlusion, hippocampal and striatal damage were reduced by approximately 50% (p<0.03) in the first experiment and by approximately 41% (p<0.002) in the second experiment. Addition of 5 ¨M of LY231617 to primary hippocampal neuronal cultures antagonized the lethal effect of hydrogen peroxide (p<0.05). Iron-dependent lipid peroxidation was also inhibited in a dose-related fashion. Conclusions The significant reduction of ischemia-induced or hydrogen peroxide-induced neuronal damage and inhibition of lipid peroxidation by LY231617 observed in this study suggest that reactive oxygen intermediates play an important role in the events leading to neuronal death after global ischemia/reperfusion.

A particulate arachidonate lipoxygenase in human blood platelets.

Abstract The microsomal fraction of human platelets contains a lipoxygenase activity in addition to the thromboxane-synthesizing activity. The enzymatic activity was stimulated by tryptophan, but inhibited by catecholamine, methemoglobin, and hydroquinones.

Biosynthesis of thromboxane B2: Assay, isolation, and properties of the enzyme system in human platelets

The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable platelet-aggregating factor and a hydrolyzed product on the thin-layer chromatography (TLC). This product was isolated on TLC, purified by silica gel column chromatography and identified by combined gas chromatography-mass spectrometry as the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9, 12L-dihydroxy-5, 10-heptadecatrienoic acid (thromboxane B2). The enzymatic activity was dependent upon methemoglobin and tryptophan as cofactors. Reduced glutathione had no effect either alone or in combination with other cofactors. Methemoglobin could be replaced by hematin or hemin; and tryptophan by 3-indolacetic acid or catecholamines. The apparent requirement for methemoglobin is due to the reductive activity of ferriprotoporphyrin IX. The reaction, however, catalyzed by the ferriprotoporphyrin IX in the thromboxane synthesizing system is different from that described for the decomposition of lipid peroxides. Certain transition metals and hydrogen donors, such as hydroquinone and ascorbate, which have been shown to stimulate the catalytic activity of ferriproroporphyrin IX in the decomposition of 15-hydroperoxy-prostaglandin E1 are inhibitors of thromboxane B2 formation. This enzyme preparation also transformed eicosa-8. 11, 14-trienoic acid to an unknown product on TLC. The enzyme system was rapidly inactivated upon incubation in the reaction mixture.

Crystalline L-Asparaginase from Escherichia coli B

L-Asparaginase has been crystallized from a partially purified extract of Escherichia coli B. The crystalline enzyme is homogeneous, as judged by analytical polyacrylamidegel electrophoresis and sedimentation behavior. This enzyme preparation is active in preventing lymphoma in mice and also has low glutaminase activity.

Fenoprofen: Inhibitor of prostaglandin synthesis

Abstract Fenoprofen, an anti-inflammatory agent, is a potent inhibitor of prostaglandin synthesis at physiological substrate concentration. I 50 s of 1 μM to 100 μM were obtained when the concentration of arachidonate was increased from 1 μM to 82 μM. In contrast, a small change in I 50 was observed with indomethacin. Kinetic studies indicated that inhibition by fenoprofen was competitive while that of indomethacin was non-competitive.

Purification and Characterization of Fatty Acid Cyclooxygenase from Human Platelets

The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGH2), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH2, that was trapped at -25 degrees C and separated on TLC at -20 degrees C. PGH2 was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE2 in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7-9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydroquinone stimulated the PGH2 formation. Metal ions, such as ZN2+ and Cd2+ inhibited the enzyme reaction at 0.1 to 1 mM. The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.

Reversal of platelet aggregation by aortic microsomes.

The microsomal fraction of dog aortas inhibited human platelet aggregation induced by arachidonic acid, ADP, or thrombin. When aortic microsomes were added to a preparation of irreversibly aggregated platelets, the aggregates dispersed after 4–6 minutes. The fact that aortic microsomes inhibit platelet aggregation induced by ADP suggests that its effect is probably on the cellular function of platelets and not in direct competition against thromboxane A2.

Enzymatic preparation of the (+),L-diastereoisomer of [methenyl-14C]-N5,N-10-methenyltetrahydrofolic acid

Abstract [Methyl-14C]-N5,N10-Methenyltetrahydrofolic acid has been prepared by the enzymatic reaction of formyltetrahydrofolate synthetase with formate and (+,−), L -tetrahydrofolate, then purified by means of column chromatography on a DEAE-cellulose column. The enzymatically synthesized material is twice as active as the chemically prepared (+,−), L -N 5 ,N 10 - methenylatetrahydrofolic acid when assayed with the N5,N10-methenyltetrahydrofolate cyclohydrolase from chicken liver. The chromatographic behavior of the enzymatically and chemically synthesized N5,N10-methenyltetrahydrofolic acid is identical. The specific rotations of these compounds were determined.

New inhibitors of dopamine β-hydroxylase

Abstract A32390A, 1,6-di-O-(2-isocyano-3-methylcrotonyl)- d -mannitol, obtained from fermentation broths of a species of Pyrenochaeta , inhibited DBH in vitro and lowered heart norepinephrine levels in vivo in rats. A32390A had less effect on adrenal catecholamine levels and did not lower brain norepinephrine levels. Associated with the inhibition of norepinephrine synthesis in vivo was a reduction of blood pressure by A32390A in DOCA hypertensive rats. Although A32390A was effective when given by either intraperitoneal or subcutaneous injection, it did not lower norepinephrine levels or blood pressure when given orally. CHMI (1-cyclohexyl-2-mercapto-imidazole) was the most active among several methimazole derivatives studied as a DBH inhibitor that reduced norepinephrine levels preferentially in brain. The initial decline in brain norepinephrine levels following CHMI injection could be used as an index of norepinephrine turnover. Lowering of brain norepinephrine levels by CHMI on the second day of proestrus in female rats prevented the surge of plasma LH levels that normally occurred on the afternoon of proestrus.

The anti-inflammatory effects of LY178002 and LY256548

LY178002 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene-4-thiazolidinone) and its N-methyl analog, LY256548, inhibit the enzymatic activity of phospholipase A2, 5-lipoxygenase and fatty acid cycloxygenase. They also inhibit leukotriene B4 production from human polymorphonuclear leukocytes stimulated with the calcium ionophore A23187. Since products of the arachidonic acid cascade have been implicated as important mediators in a variety of inflammatory diseases including arthritis, LY178002 and LY256548 were studied in the Freunds Complete Adjuvant-Induced Arthritis (FCA) model in rats. The compounds were administered orally and inhibition of bone damage and paw swelling was assessed of both the injected and uninjected paws. At 50 mg/kg LY178002 inhibited soft tissue swelling in the uninjected paw by 81% while LY256548 exhibited 57% inhibition. Bone damage was also significantly inhibited by both compounds. A dose response was conducted. The minimum effective dose for LY178002 was 10 mg/kg p.o. In the established FCA model LY178002 at 50 mg/kg p.o. inhibited the uninjected paw swelling by 75% while LY256548 did not show this level of activity. These results suggest that LY178002 and LY256548 may be useful in the treatment of arthritis.