Biomedical subjects
Publications and source records attributed to S Toki.
A novel compound, PS-990, which induces differentiation of neuroblastoma cells, was isolated from the culture broth of a fungus, Acremonium sp. KY12702. PS-990 inhibited brain calcium calmodulin-dependent cyclic nucleotide phosphodiesterase with an IC50 value of 3 micrograms/ml, and markedly induced neurite extension of mouse neuroblastoma, Neuro2A, at concentrations ranging from 10 to 30 micrograms ml.
ES-242-1, a novel bioxanthracene of microbial origin, blocked glutamate-induced neuronal death in a dose-dependent manner at concentrations ranging from 0.01 to 1.0 microM, but not the neuronal death caused by kainic acid or quisqualic acid. ES-242-1 also prevented cell death induced by 2,4-methanoglutamate, which is a specific agonist for the NMDA receptor. ES-242-1 showed protective effects in cultured neurons prepared from cerebellum and septum as it did in cultured hippocampal neurons but to different extents. There was a positive correlation between the potencies of ES-242s as inhibitors of ligand binding to the NMDA receptor and as inhibitors of neuronal death. Hypoxic treatment for 4 h under 95% N2 and 5% CO2 caused neuronal death of the cultured hippocampal neurons. Again, ES-242-1 at 1.0 microM was effective to protect neurons against hypoxic injury. ES-242 compounds are new chemical entities possessing neuroprotective properties useful in the treatment of diseases involving glutamate toxicity.
1. In the presence of glutathione under physiological conditions, 3'-oxohexobarbital was non-enzymically converted to 1,5-dimethylbarbituric acid and a cyclohexenone-glutathione adduct. 2. The two reaction products were characterized by mass spectrometry, 1H- and 13C-n.m.r. spectrometry, and UV spectral analyses. 3. 1,5-Dimethylbarbituric acid was excreted in urine of rat given hexobarbital, 3'-oxohexobarbital, or 1',2'-epoxyhexobarbital, and accounted for 13.4, 14.5 and 4.7% of dose, respectively. 4. The cyclohexenone-glutathione adduct, a novel metabolite of hexobarbital, was excreted in the bile of rat given hexobarbital. 5. The route of 1,5-dimethylbarbituric acid formation via 3'-oxohexobarbital in the metabolism of hexobarbital was discussed in comparison with the epoxide-diol pathway.
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ES-242-1 approximately 5 are novel microbial bioxanthracenes which do not contain nitrogen. The ES-242s inhibited the binding of [3H]TCP and [3H]CGS19755 to the N-methyl-D-aspartate (NMDA) receptor complex. They had no effect on the binding of the specific ligands for the non-NMDA receptor. The biochemical and pharmacological properties of ES-242-1 were fully examined since it is the most potent of the five compounds. ES-242-1 is highly specific for the NMDA receptor; it has no effect on other receptors. Kinetic analyses indicated that ES-242-1 inhibited the binding of [3H]TCP and [3H]CGS19755 in a competitive manner, respectively, suggesting that ES-242-1 interacts with both the transmitter recognition site and the channel domain. ES-242-1 selectively inhibited NMDA-induced Ca2+ influx in primary cultures of mouse hippocampal neurons. ES-242-1 also specifically blocked the increase in cyclic GMP accumulation induced by NMDA or L-glutamate in rat cerebellar slices. In a concentration range of 0.1-1.0 microM, ES-242-1 was as potent as MK-801 in preventing glutamate-induced neurotoxicity in primary cultures of mouse hippocampal neurons. These results show that ES-242-1 is a potent and specific antagonist for the NMDA receptor. The antagonistic properties of the ES-242s appear to be due to a novel mechanism of action at the NMDA receptor.
HS-142-1 is a novel non-peptide antagonist for atrial natriuretic peptide (ANP) receptor. The effect of HS-142-1 on the cyclic GMP production elicited by natriuretic peptides in neuronal cell lines, PC12 and NG108-15 was examined. Natriuretic peptides such as ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) enhanced cyclic GMP production in a dose-dependent manner. HS-142-1 inhibited cyclic GMP accumulation elicited by natriuretic peptides in a dose-dependent fashion in both cells. The results suggest that HS-142-1 will be an important tool for identification and understanding of the mechanisms by which natriuretic peptides act in nervous systems.
1. The hydroxyl radical-mediated conversion of morphine to morphinone (MO) was examined as an alternative to the enzymic reaction. 2. Hydroxyl radicals were generated by autoxidation of ascorbate in the presence of iron and EDTA. This system oxidized morphine to MO which was identified by h.p.l.c. and t.l.c. The reaction was dependent on the concentration of added Fe2+ and required the addition of ascorbate when Fe3+ was used. 3. Catalase inhibited production of MO whereas superoxide dismutase (SOD) had no effect. Addition of a large amount of H2O2 to the system resulted in a significant decrease in production of MO. No MO production was initiated by H2O2 itself. The oxidation of morphine was inhibited by typical hydroxyl radical-scavenging agents. These results indicate that morphine undergoes oxidation to MO by hydroxyl radical.
Verticillium sp. SPC-15898 was found to produce novel metabolites, designated ES-242-2-(-)8, which were structurally related to ES-242-1. These compounds were isolated from the culture broth and the physico-chemical and biochemical properties were examined. ES-242-2-(-)8 inhibited [3H]thienyl cyclohexypiperidine ([3H]TCP) binding to rat crude synaptic membranes (CSM) with IC50 values of 0.116, 2.9, ca. 2.9, 25.3, 1.0, 59, 24, and 13 microM, respectively. None of these compounds showed inhibitory effects against the binding of [3H]kainate to its receptor, which is another subtype of the excitatory amino acid receptor.
A novel compound, ES-242-1, which binds to a site on N-methyl-D-aspartate (NMDA) receptor that is coupled to the channel domain, was isolated from the culture broth of a fungus, Verticillium sp. SPC-15898. ES-242-1 inhibited the [3H]thienyl cyclohexylpiperidine ([3H]TCP) binding to rat crude synaptic membrane fractions with an IC50 value of 116 nM, but did not inhibit the [3H]kainate binding to its receptor, which is another subtype of the excitatory amino acid receptor.
Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.
The biliary excretion of morphine and its metabolites by guinea pigs after s.c. injection of morphine (25 mg/kg) was determined by high-performance liquid chromatography procedures. The amounts of morphinone (MO), morphinone-glutathione adduct (MO-GSH), morphine and morphine-3-glucuronide excreted over a 4-hr period were 1.27, 9.35, 1.13 and 7.54% of the administeral dose, respectively. In addition, trace amounts of morphine-GSH adduct and morphinone-cysteine adduct, derived from MO-GSH, were detected. Although MO-GSH formation from MO proceeded nonenzymatically with a relatively high rate, GSH S-transferases participated in the reaction, especially at lower GSH concentrations. Pretreatment of the animals with diethyl maleate and lithocholic acid-3-sulfate decreased the total biliary excretion of MO. On the other hand, pretreatment with naloxone increased the excretion of total MO. In vitro experiments using liver cytosolic preparations showed that lithocholic acid-3-sulfate inhibited the enzymatic formation of MO from morphine and of MO-GSH from MO. The effects of naloxone on MO formation in vitro were more complex. A reaction was stimulated at low and suppressed at higher concentrations. The results indicated that MO-GSH formation from morphine is inhibited by lithocholic acid-3-sulfate through actions on both morphine 6-dehydrogenase and GSH S-transferases. Low levels of naloxone were found to stimulate the morphine 6-dehydrogenase-mediated dehydrogenation of morphine to MO.
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A serine hydroxymethyltransferase was purified to complete homogeneity from a serine-producing obligate methylotroph, Hyphomicrobium methylovorum GM2. The enzyme has a molecular mass of about 98 kDa and consists of two subunits of identical molecular mass. The holoenzyme exhibits absorption maxima at 280 nm, 340 nm and 415 nm in potassium phosphate buffer, pH 7.3, the last of which shifts with a change in pH (6.0-7.5) and contains 2 mol pyridoxal phosphate/mol enzyme. The holoenzyme is converted to the apoenzyme on incubation with phenylhydrazine and reconstituted on the addition of pyridoxal phosphate. The enzyme activity was inhibited on the addition of several sulfhydryl-modifying reagents and then recovered with 2-mercaptoethanol. One sulfhydryl group per subunit was found to be responsible for the activity. Isoelectric focusing showed that the enzyme has a pI of 5.6. The Km values for glycine, L-serine and DL-beta-phenylserine are 0.046 mM, 0.15 mM and 33 mM respectively.
Elution profiles of guinea-pig liver naloxone reductase and morphine 6-dehydrogenase on Matrex green A, Sephadex G-100 and DEAE-cellulose (DE32) column chromatography used sequentially in the purification procedure were identical. The ratios of the two enzyme activities were almost constant throughout all the purification steps. The two enzymes were similarly more stable at pH 6.0 than at pH 8.0 on storage at 4 degrees. The reversible inactivation of the two enzymes by the removal of 2-mercaptoethanol from the enzyme solution was the same. Inhibitory effects of lithocholic acid, CuSO4, quercitrin, phenylarsine oxide, and prostaglandin E1 on the two enzymes were almost the same. These results indicated that naloxone reductase is identical to morphine 6-dehydrogenase in the guinea-pig liver. For the reduction of naloxone, the enzyme utilized either NADPH or NADH as cofactor, and pH optima were 6.8 with NADPH and 6.2 with NADH. The Km values for NADPH and NADH were 6.5 and 2.2 microM respectively. The Vmax values for naloxone were 1.2 units/mg protein with NADPH and 0.5 unit/mg protein with NADH. The Km values for naloxone were 0.27 mM with NADPH and 0.44 mM with NADH. The reaction product formed by the enzyme was identified as 6 alpha-naloxol by thin-layer and gas-liquid chromatographic analyses. Accordingly, it is clear that the enzyme catalyzes the stereospecific reduction of naloxone to form the 6 alpha-hydroxyl congener.
Optimal culture conditions of a methylotrophic Hyphomicrobium methylovorum and improved purification of serine hydroxymethyltransferase from the bacterium were established for the large-scale preparation of the enzyme. The first crystalline serine hydroxymethyltransferase from the microbial source was obtained in the apo form and found to be homogeneous. Amino acid analysis revealed that the enzyme had higher value per subunit for acidic and neutral amino acids than that from rabbit liver. The carboxy-terminal amino acid analysis suggested the sequence -Ile-Ala-Tyr.
The ability of sulfhydryl compounds to provide protection against the acute toxicity of codeinone, a toxic metabolite of codeine, was investigated in mice. Subcutaneous administration of codeinone produced a slight reduction in hepatic glutathione concentration. Pretreatment of the mice with glutathione or cysteine significantly increased the survival rate for mice given a lethal dose of codeinone (10 mg/kg). The lethality of codeine was lowered by naloxone, whereas that of codeinone was not blocked by naloxone. The strychnine-like convulsant action of codeinone could be prevented by phenobarbital pretreatment. Glutathione pretreatment reduced the amounts of radioactivity in tissues of mice injected with [N-methyl-3-H]codeinone. A possible explanation for these observations is that glutathione reacts in vivo with codeinone and plays a role as a scavenger of this compound. This assumption is supported by the observation that codeinone reacts non-enzymatically with glutathione under physiological conditions.