[Analysis of developmental pattern of neurotransmitters and receptors using primary cultured neurons: with special reference to GABAergic neurons].
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Animals↗
Biomedical subjects
S Ohkuma
Publications and source records attributed to S Ohkuma.
Peroxidized lipids isolated by HPLC from atherosclerotic aorta.
Conjugated dienes of fatty acids were detected in cholesterol esters in lipid droplets prepared from atherosclerotic aorta of WHHL rabbits. Two peroxidized fatty acids were recovered by reverse phase HPLC. These peroxidized fatty acids showed maximum absorption at 233 nm, suggesting that they were peroxidized lipids containing cis-trans conjugated diene, and one of them was shown to be 13-hydroxy octadecadienoic acid.
Animals↗
Regulatory role of cysteine dioxygenase in cerebral biosynthesis of taurine. Analysis using cerebellum from 3-acetylpyridine-treated rat.
The effect of 3-acetylpyridine (3-AP) administration on the biosynthesis of taurine in the rat brain has been studied. Treatment with 3-AP induced a significant decrease in the cerebellar contents of taurine and its metabolic precursors, cysteine sulfinic acid (CSA) and cysteic acid (CA), as well as a selective degeneration of climbing fibers in the molecular layer of the cerebellum. It was found that the activity of cerebral cysteine dioxygenase, the enzyme catalyzing the formation of CSA from cysteine, consisted of two systems with low and high Km values. The 3-AP-induced attenuation of cysteine dioxygenase activity with a low Km value was noted only in the cerebellum, while that with a high Km value was detected not only in the cerebellum but also in other brain areas such as the medulla oblongata, striatum and cerebral cortex. In contrast, no alteration in the activity of cysteine sulfinic acid decarboxylase (CSD) was observed in any brain areas examined following the administration of 3-AP. Furthermore, it was found that essentially no cystamine as well as a very low activity of cysteamine dioxygenase is present in the brain. The present results suggest that taurine in the brain is synthesized from cysteine, mainly by the CSA and CA pathways, and the observed decline of cerebellar taurine in 3-AP-treated rats may be due to an attenuation of the biosynthesis, possibly at the step of cysteine dioxygenase. A possible regulatory role of cysteine dioxygenase with a low Km value in the biosynthesis of cerebral taurine is also suggested.
Animals↗
Biliary excretion of novel pneumotoxic metabolites of the pyrrolizidine alkaloid, monocrotaline.
Perfusion of the pyrrolizidine alkaloid, monocrotaline, through the isolated rat liver resulted in the appearance of Ehrlich-positive metabolites in both the perfusate and bile. Livers from male rats released greater quantities of metabolite into both bile and perfusate. Metabolite release was stimulated by pretreatment with phenobarbital. Livers of phenobarbital-pretreated male rats perfused with 30 microM monocrotaline produced biliary metabolite concentrations in excess of 5 mM. Metabolite release was inhibited by anoxic perfusion, low temperature and pretreatment with SKF-525A. Above 150 microM monocrotaline, bile became the predominant route of excretion. On perfusion through the isolated lung of the rat, bile and perfusate metabolites were equally effective in inhibiting serotonin transport. A single Ehrlich-positive peak was obtained on silica gel column chromatography of bile containing metabolite. Mass spectrometry revealed the major component of this peak to have a molecular weight of 281, indicating a novel pyrrole metabolite in which the esterifying acid present in monocrotaline, monocrotalic acid, had been partially degraded. This compound mimics the pneumotoxic action of monocrotaline given in vivo, and its availability should prove a valuable tool in the elucidation of the mechanism of pyrrolizidine toxicity.
Animals↗
Hepatic venocclusive disease associated with the consumption of pyrrolizidine-containing dietary supplements.
Venocclusive disease, a form of Budd-Chiari syndrome, was diagnosed in a 49-yr-old woman. The patient had portal hypertension associated with obliteration of the smaller hepatic venules. A liver biopsy specimen showed centrilobular necrosis and congestion. Analysis of food supplements the woman regularly consumed showed the presence of pyrrolizidine alkaloids. The major source was a powder purporting to contain ground comfrey root (Symphytum sp). We calculated that during the 6 mo before the woman was hospitalized, she had consumed a minimum of 85 mg of pyrrolizidine alkaloids (15 micrograms/kg body wt X day). The clinical and analytic findings were consistent with chronic pyrrolizidine intoxication, indicating that low-level, chronic exposure to such alkaloids can cause venocclusive disease.
Budd-Chiari Syndrome↗
Positional specificity of lysosomal acid lipase purified from rabbit liver.
The hydrolysis of tri-, di-, and monooleoylglycerols by highly purified lysosomal acid lipase from rabbit liver (Imanaka, T., et al. (1984) J. Biochem. 96, 1089-1101) was examined. The degradative products were separated by thin layer chromatography on silicic acid impregnated with boric acid. Trioleoylglycerol was hydrolyzed with intermediate accumulation of 1,2(2,3)-dioleoylglycerol and 2-monooleoylglycerol, but no detectable production of the 1,3-isomer. 1,2(2,3)-Dioleoylglycerol was also hydrolyzed with the intermediate accumulation of 2-monooleoylglycerol. The 1(3)-isomer of monooleoylglycerol was hydrolyzed exclusively, with no hydrolysis of the 2-isomer. These results indicate that the enzyme shows preference for 1(3)-ester bonds and that the main lipolytic reaction sequence catalyzed is triacylglycerol---1,2(2,3)-diacylglycerol---2-monoacylglycerol.
Animals↗
Effect of phospholipids on the hydrolysis of cholesterol oleate liquid crystals by lysosomal acid lipase.
Cholesterol oleate liquid crystals were prepared in vitro as a model of lipid droplets accumulated in the atheromatous aorta. The hydrolysis of cholesterol oleate crystals by lysosomal acid lipase was examined in the presence and absence of various phospholipids. Phosphatidylserine markedly stimulated the hydrolysis of cholesterol oleate liquid crystals (20 times the basal value); it increased the Vmax value about 15 times and decreased the Km value to 1/20 times the basal value. The polar head group and the acyl chains of phosphatidylserine were required for its stimulation of hydrolysis.
Animals↗
[Cellular responses and the H+ pump in the vacuolar system].
Intracellular acidic compartments are now recognized to be ubiquitous in the vacuolar system of eukaryotic cells, where the unique ATP-dependent H+ pump (s) functions to maintain their internal low pH. Furthermore, various basic substances and acidic ionophores have been found to raise the internal pH of these compartments to neutrality. These chemicals, on the other hand, have been found to affect various biological reactions in multicellular organisms, including those involved in intercellular communication. The properties of the H+ pump of the vacuolar system and its role in cellular responses are described.
Adenosine Triphosphate↗
Effect of esterastin, an acid lipase inhibitor, on the free and esterified cholesterol contents of cultured aortic smooth muscle cells treated with LDL and cholesterol ester liquid crystals.
The effects of esterastin, an acid lipase inhibitor, on the free and esterified cholesterol contents of cultured smooth muscle cells from pig aorta were examined. The post-nuclear supernatant fraction of the cell homogenate showed maximum acid cholesterol esterase activity at pH 4.5, and 50% of this activity was inhibited by 0.31 microM esterastin. During a 48 h incubation with esterastin, the esterified cholesterol content of the cells increased to about 13 times that of control cells in the presence of low density lipoprotein and to 7 times that of control cells in the presence of cholesterol oleate liquid crystals. The ratio of esterified to free cholesterol also increased to about 5 times the control value in both conditions.
Animals↗
Alteration of cerebral biosynthesis of taurine in spontaneously hypertensive and 3-acetylpyridine intoxicated rats.
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Animals↗
Subcellular localization of acid lipase in rat liver.
The intracellular localization of acid lipase was examined by several subcellular fractionation techniques. Upon differential centrifugation, acid lipase was detected mainly in the lysosomal fraction. When linear sucrose density gradient centrifugation was used, the mean density of acid lipase was similar to that of lysosomal marker enzymes. After Triton-treatment of rats, however, acid lipase was recovered at a density of 1.10-1.16, which is more than that of tritosomes, when tissues were homogenized with a Potter-Elvehjem homogenizer. But by homogenizing the liver from these rats by pressing it through a stainless steel net, acid lipase was mainly recovered at the same density as that of tritosomes. These results indicate that acid lipase is localized mainly in lysosomes in rat liver.
Animals↗
Characteristics of taurine transport in rat hepatocytes in primary culture.
Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.
Animals↗
Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes.
Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.
Animals↗
Determination of cystamine by high-performance liquid chromatography.
A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.
Animals↗
Proton translocating ATPase in lysosomal membrane ghosts. Evidence that alkaline Mg2+-ATPase acts as a proton pump.
Membrane ghosts were prepared from purified lysosomes (tritosomes) of rat liver by hypo-osmotic treatment. Mg2+-ATP-driven acidification was observed in the membrane ghosts using acridine orange as a fluorescent probe of the transmembrane pH gradient (delta pH). Its properties were the same as those of intact lysosomes reported previously (Ohkuma, S., Moriyama, Y., & Takano, T. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762; Moriyama, Y., Takano, T., & Ohkuma, S. (1982) J. Biochem. 92, 1333-1336). The H+-pump was found to be electrogenic with use of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol as a fluorescent membrane potential probe. Alkaline Mg2+-ATPase activity was also identified on the membranes. It showed a pH maximum of pH 8.0-8.5, a Km value for ATP of 0.36 mM and a Vmax of 0.41 units/mg protein at 30 degrees C. Its activity was inhibited by dicyclohexylcarbodiimide, tri-n-butyltin, azide and ADP, but not by ouabain or vanadate. It differed from mitochondrial F1F0-ATPase in sensitivities to N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and oligomycin. Since this alkaline Mg2+-ATPase activity is very similar to the H+-pump activity in its requirement for divalent cations, substrate specificity and sensitivities to various chemicals, it may act as a proton translocase (H+-pump). Possible mechanisms of action of some chemicals, such as 4-acetamide-4'-isothiocyanatostilbene-2,2'-disulfonic acid, that inhibited the H+-pump but not the alkaline Mg2+-ATPase, are discussed.
4-Chloro-7-nitrobenzofurazan↗
Solubilization and reconstitution of a lysosomal H+-pump.
H+-ATPase was solubilized from the lysosomal membranes of rat liver with lysophosphatidylcholine. The detergent was removed with Biobeads SM2, and the proteoliposomes were prepared by rapid dilution of a mixture of the solubilized ATPase, octyl-beta-D-glucopyranoside and liposomes. The proteoliposomes exhibited ATP-dependent fluorescence quenching of quinacrine and bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol. These results suggested that the electrogenic lysosomal H+-pump was solubilized and reconstituted into proteoliposomes.
Animals↗
Characterization of lysosomal acid lipase purified from rabbit liver.
Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.
Animals↗
Correlation between two active forms of valyl-tRNA synthetase and aminoacylation levels of two valine isoaccepting tRNA species in the early stages of sporulation of Bacillus subtilis.
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Amino Acyl-tRNA Synthetases↗