Studies on the metabolic pathway of the acetyl group for acetylcholine synthesis

Glucose and pyruvate are the most effective precursors of the acetyl moiety of acetylcholine in mammalian brain; the metabolic intermediates between pyruvate and acetylcholine, however, are unknown. The following data suggest that citrate is not the sole intermediate of the acetyl group for acetylcholine synthesis in rat brain slices or synaptosomes: (1) 2.5 mM (?)-hydroxycitrate decreased acetylcholine synthesis from [U-¹⁴C]glucose by only 25 per cent; (2) inhibition of citrate transport out of mitochondria by n-butylmalonate or 1,2,3-benzenetricarboxylate variably affected acetylcholine synthesis; and (3) high concentrations of nonradioactive citrate decreased the synthesis of acetylcholine but did not decrease the specific activity of the acetylcholine synthesized from [U-¹⁴C]glucose. even though the uptake of citrate into the synaptosomes under these experimental conditions was approximately five times greater than the uptake of glucose. Other possible acetyl donors altered acetylcholine synthesis. Acetylcarnitine stimulated synthesis in brain slices, and carnitine stimulated synthesis by synaptosomes.The specificactivity of the acetylcholine synthesized from [U-^14Cglucose by synaptosomes was decreased by N-acetyl-l-aspartate (10mM), acetyl CoA (1 mM), and acetyl phosphate (10mM) which is consistent with these compounds acting as direct acetyl donors. Acetate (10 mM) did not affect either the amount or specific activity of the acetylcholine synthesized. Further evidence of compartmentation of cytoplasmic acetyl CoA is presented. The cytoplasmic acetyl CoA for acetylcholine synthesis is distinguishable from the cytoplasmic acetyl CoA for lipid synthesis. (?)-Hydroxycitrate inhibited acetylcholine synthesis without inhibiting lipid synthesis from [U-¹⁴C]glucose. However, when 3-hydroxy[3-¹⁴C]butyrate was used as substrate, (?)-hydroxycitrate inhibited incorporation into lipids twice as much as incorporation into acetylcholine. [U-¹⁴C]Glucose metabolism by infant brain slices was more sensitive than adult brain slices to (?)-hydroxycitrate. However, the response to the other compounds which interfere with citrate metabolism was similar in slices from adult and infant brains.     

Glutathione S-transferases in nitrogen mustard-resistant and -sensitive cell lines

Tumor cell resistance to alkylating agents was studied by examining Walker 256 rat mammary carcinoma cells differentially sensitive to nitrogen mustards. A resistant subpopulation (WR) was selected by exposure to chlorambucil. WR cells showed approximately a 15-fold resistance to the cytotoxic effects of nitrogen mustards and elevated glutathione S-transferase (GST) activity when compared to the sensitive parent cell line (WS). To extend these findings, the GSTs from WR and WS were purified by affinity chromatography on S-hexylglutathione coupled to epoxy-activated agarose. Substrate specificity experiments using purified GSTs demonstrated different profiles of enzyme activity for WR and WS and suggested differential isoenzyme expression in these two cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that the major GST present in both WR and WS was a 26,000-Da subunit that was immunologically distinct from the rat liver GSTs. This GST subunit cross-reacted with antibodies against anionic human placental GST. In addition, three GST forms common to rat liver (29,500, 28,500 and 27,500 molecular weight) were also identified. Overexpression of the 29,500-Da protein was observed in WR cells. These data suggest that differential expression of GST subunits may contribute to the nitrogen mustard-resistant phenotype.     

Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WRr and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.     

Effects of adrenochrome and sodium perborate on isolated fat cell metabolism

The effects of adrenochrome, epinephrine and sodium perborate on glucose oxidation in isolated white fat cells were studied. Adrenochrome and sodium perborate were found to preferentially stimulate oxidation of [-1-¹⁴C]glucose while epinephrine stimulated [-6-¹⁴C]glucose oxidation. The stimulation by adrenochrome, epinephrine and sodium perborate of glucose oxidation was not appreciably effected by treating fat cells with trypsin. Treatment of the albumin used with the metal chelator 1,10-phenanthroline decreased but did not abolish the effectiveness of these agents.Epinephrine in the presence of bovine serum albumin was converted to a pink-colored product which exhibited an absorbance maximum at 300 nm. However, it is unlikely that the effects of epinephrine on D-[-1-¹⁴C]glucose oxidation are the result of its conversion to adrenochrome.Adrenochrome was also found to cause an increase in the accumulation of cyclic AMP over a 10-min incubation period. No effects of adrenochrome were observed on the activity of adenylate cyclase stimulation by glucagon and epinephrine, but adrenochrome did inhibit the stimulation of cyclase activity by fluoride ion. Adrenochrome was active as an inhibitor of phosphodiesterase present in fat cells.     

Purification and acetylation of protein X subunit of pyruvate dehydrogenase complex (PDC) from bovine kidney

Protein X is one of the subunits of pyruvate dehydrogenase complex. The biological role of this protein has not been fully elucidated, mainly because of the difficulty in its dissociation from the tightly bound dihydrolipoamide acetyltransferase-protein X subcomplex. We have found that the detachment of protein X from acetyltransferase subunit can be easily accomplished by the cycles of freezing and thawing process. Several lines of evidence including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis and acetylation with [2-¹⁴C] pyruvate confirmed that the purified protein is protein X. The purified intact form of protein X was acetylated by [2-¹⁴C] pyruvate in the presence of pyruvate dehydrogenase subunit. The acetylation efficiency of this protein was lower than that of acetyltransferase and was not affected by the presence of acetyltransferase.     

FcεRI-stimulated Ca2+-dependent secretion from rat basophilic leukemia (RBL-2H3) cells permeabilized with Staphylococcal α-toxin: FcεRI-operated signals are not mimicked by the actions of GTPγS

1. RBL-2H3 cells permeabilized with α-toxin responded to dinitrophenol (30–40 mol/mol)-conjugated human serum albumin, as antigen, to secrete [¹⁴C]serotonin in the micromolar range of free Ca²⁺.2. Calcium ion alone did not cause substantial secretion.3. Guanosine 5′-O-(3-thiotriphosphate) (GTP_γS) (100 μM) in combination with Ca²⁺ produced only negligible [¹⁴C]serotonin secretion.4. GTP_γS, in the presence of cytochalasin D, caused optimal secretion of [¹⁴C]serotonin in a Ca²⁺-dependent manner.     

Adenosine 5'-triphosphate-citrate lyase activity in mammalian spermatozoa: A new radiometric method and characterization of the enzyme in spermatozoa

A simple radiochemical method was developed for determining the ATP-citrate lyase activity in mammalian spermatozoa. The determination of enzyme activity was followed by the measurement of the incorporation of the [1-¹⁴C]acetyl group from [1,5-¹⁴C]citrate into [1-¹⁴C]acetylcoenzyme A (ACoA). Separation of ¹⁴C-labeled ACoA from the reactants and their products was achieved by rapid anion exchange chromatography. The optimum pH was 6.4 for rat spermatozoal ATP-citrate lyase. The activity was not altered by dithiothreitol. MgCl₂ (l0mM) caused a 50 per cent inhibition in the enzyme activity. ATP-citrate lyase activities in rat and human spermatozoa were 154 ± 14 and 90 ± 12 nmoles of ACoA formed/mg of protein/5 min. Citrate may serve as an acetyl source for acetylcholine formation by spermatozoa.     

Effect of phenobarbital on the incorporation of 3H or 14C labeled precursors into fatty acids

The incorporation of various ³H or ¹⁴C labeled precursors into hepatic fatty acids was studied in control and phenobarbital-treated rats. In vitro, phenobarbital had no effect on fatty acid synthesis from the tritiated precursors ³H₂O, 1-³H glucose, 2-³H lactate, 2,3-³H succinate and 2-³H acetyl CoA or from the ¹⁴C labeled precursors 1-¹⁴C acetate and 1,3-¹⁴C malonyl CoA in liver supernatant or supernatant + microsomes preparations. In vivo, phenobarbital stimulated the incorporation of 1-¹⁴C acetate, ³H₂O, 2-³H lactate and 2,3-³H succinate but had no stimulatory effect on the incorporation of 1-³H glucose. The activities of lactic dehydrogenase, glucose 6-phosphate dehydrogenase and succinate cytochrome c reductase were not modified by administration of phenobarbital but that of NADPH-cytochrome c reductase was increased. These results indicate that the NADH and NADPH pools are not quite in equilibrium, and that the endoplasmic reticulum probably competes with the cytoplasm for NADPH utilization and thus may play a part in the regulation of fatty acid synthesis. The increased incorporation of ¹⁴C precursors observed in vivo in phenobarbital-treated rats was not due to stimulation of the synthesis of the key enzymes of fatty acid synthesis but could be related to an activation of these enzymes which, in vivo, are probably fixed on the endoplasmic reticulum.     

Syntheses of [2-14C]penem antibacterials; (FCE 22101 and FCE 22891)

The synthesis of FCE 22101 (sodium (5R,6S)-6-[(1R)-hydroxyethyl]-2-carbamoyloxymethylpenem-3-carboxylate) labelled with carbon-14 in the 2-position of the penem system ring was performed in eight steps, using sodium salt of [1-¹⁴C]glycollic acid 1 as the labelled starting material. The final product, penem [2-¹⁴C]FCE 22101 11, was obtained in an overall radiochemical yield of 21%, 98% radiochemically pure and with a specific activity of 641 MBq/mmol (17.3 mCi/mmol). The acetoxymethyl ester FCE 22891 12 was prepared by condensation of 11 with bromomethyl acetate, with a yield of 41%.     

Selection of nitrogen mustard resistance in a rat tumor cell line results in loss of guanine-O6-alkyl transferase activity

Cell killing, DNA-interstrand crosslinks, and DNA-protein crosslinks were assayed in nitrogen mustard-resistant Walker 256 carcinoma (WR) cells and the parent cell line (WS) after treatment with 5-[3-(2-chloroethyl)-1-triazenyl]imidazo-4-carboxamide (MCTIC). The WR cells, which also express collateral sensitivity to chloroethylnitrosoureas, were approximately twice as sensitive to the cytotoxic effects of MCTIC as were WS cells. Following treatment with 100 microM MCTIC, there was a rapid accumulation of both DNA-interstrand and DNA-protein crosslinks in the WR cell line, which reached a maximum at 6 and 12 hr, respectively. There was considerably less crosslinking in the WS cells and both cell lines were proficient in repairing most of the crosslinks by 24 hr. Measurement of guanine-O6-alkyl transferase activity showed the enzyme to be present in WS but not in WR cells. These data indicate that the collateral sensitivity of nitrogen mustard-resistant WR cells to chloroethylating drugs is in part due to the loss of guanine-O6-alkyl transferase activity which is present in the parent line.     

Tritium- und 14C-markiertes Dithranol-1,8,9-triacetat

Tritium- and ¹⁴C-Labelled Dithranol 1,8,9-Triacetate Dithranol (1) is converted to 1,8,9-[¹⁴C]-triacetoxyanthracene (2) by treatment with [1-¹⁴C]-acetic anhydride. 1,8,9-Triacetoxy-[³H]-anthracene is produced by reduction of [³H]-1,8-dihydroxy–9,10-anthraquinone (6) followed by acetylation.     

Uptake and retention of adriamycin and daunorubicin by sensitive and anthracycline-resistant sublines of P388 leukemia.

Sublines of P388 leukemia completely resistant to adriamycin (P388/ADR) or daunorubicin (P388/DAU) in vivo were studied in vitro. These sublines were more resistant to the cytotoxic effects of adriamycin (800-fold relative to sensitive parental cell line, P388/S) than to daunorubicin (18-fold for P388/ADR and 56-fold for P388/DAU). When the effects of the drugs on thymidine incorporation were compared in vitro in sensitive and resistant cells, it was observed that slightly higher levels of the drugs were required to inhibit nucleic acid synthesis in the resistant cells. The shift in inhibitory concentration was much less than the shift in cytotoxic concentration, particularly for adriamycin. The uptake and efflux of [G-³H]daunorubicin and [14-¹⁴C]adriamycin were studied. At low concentrations uptake of both drugs was impaired in the resistant sublines, whereas, at high concentrations a difference in uptake between sensitive and resistant cells was not evident. Resistance did not appear to be related to the difference in the rate of uptake. A markedly enhanced efflux of the drugs from the resistant cells was observed which correlated well with the difference in sensitivity of the sublines to adriamycin and daunorubicin. Enhancing the uptake of adriamycin by increasing the pH of the incubation medium and thereby increasing the proportion of non-ionized drug available for diffusion into the cells or by modifiying the cell membrane by the addition of Tween 80 failed to reverse resistance. The binding of daunorubicin to isolated nuclei from P388/S and P388/ADR cells was essentially similar. It is concluded that these anthracycline-resistant cell lines are resistant by virtue of decreased retention of the drugs.     

Effect of 2,5-Hexanedione on lipid biosynthesis in sciatic nerve and brain of the rat

Distal axonopathy induced by n-hexane and methyl n-butyl ketone has been attributed to a common metabolite, 2,5-hexanedione. Since altered lipid metabolism is frequently associated with neuropathy, the effects of 2,5-hexanedione on lipid biosynthesis from [1-¹⁴C]acetate in sciatic nerve and brain of rats given 1% 2,5-hexanedione in drinking water have been studied, in vitro. Clinical signs of neuropathy appeared after 6 weeks. Loss of body weight induced by 2,5-hexanedione was similar to that observed in pair-fed control rats. Compared to nerves from pair-fed controls, nerves from rats fed 2,5-hexanedione exhibited decreased incorporation of [1-¹⁴C]acetate into triacylglycerols (32%), total sterols + diacylglycerols (54%), digitonin-precipitable sterols (55%), squalene (55%), and ubiquinone (43%). Incorporation of [1-¹⁴C]acetate into phospholipids, fatty acids, and cholesteryl esters was similar in nerves of 2,5-hexanedione-treated rats and pair-fed controls. In brain, incorporation of [1-¹⁴C]acetate into lipids was similar in 2,5-hexanedione-treated and pair-fed control rats, except into the fatty acid fraction which was significantly decreased by 11%. The data support the hypothesis that lipid metabolism, in particular sterol metabolism, is altered in hexacarbon-induced distal axonopathy.     

核素掺入研究布替萘芬对真菌麦角甾醇生物合成的影响

目的 研究抗真菌药物布替萘芬对真菌麦角甾醇生物合成的影响。方法 通过真菌活细胞[1-¹⁴C]乙酸钠掺入和离细胞液[2-¹⁴C]甲羟戊酸掺入实验,考察布替萘芬对真菌麦角甾醇生物合成的影响。结果 布替萘芬能使真菌中麦角甾醇的合成减少,而增加角鲨烯的含量,两种方法测得布替萘芬对真菌细胞麦角甾醇生物合成的IC₅₀分别为136.19 nmol/L和203.15 nmol/L。结论 布替萘芬为角鲨烯环氧化酶抑制剂,核素掺入法能定量考察布替萘芬对角鲨烯环氧化酶的抑制活性,且效果优于薄层色谱扫描法。     

Carrier-Mediated Transport of Monocarboxylic Acids in Primary Cultured Epithelial Cells from Rabbit Oral Mucosa

Purpose. Using primary cultured rabbit oral mucosal epithelial cells (ROEpi), we investigated whether carrier-mediated drug absorption via the oral mucosal route occurs. Methods. Oral mucosal epithelial cells were isolated from rabbit buccal mucosa and cultured on tissue culture plates. When the cells reached confluence, drug uptake experiments were performed. [¹⁴C]Benzoic acid or [¹⁴C] acetic acid was used as a marker for monocarboxylic acid carrier-mediated transport. Results. The uptake of [¹⁴C]benzoic acid by ROEpi occurred at a much lower rate at 4°C than at 37°C. The metabolic inhibitors, sodium azide and 2,4-dinitrophenol, significantly inhibited the uptake of [^l4C]benzoic acid by ROEpi. Various monocarboxylic acids inhibited the uptake of [¹⁴C]benzoic acid or [¹⁴C]acetic acid by ROEpi, whereas dicarboxylic acids did not affect the uptake. Kinetic analysis using Lineweaver-Burk plots revealed that acetic acid competitively inhibited the uptake of [^l4C]benzoic acid, and benzoic acid competitively inhibited the uptake of [^l4C]acetic acid by ROEpi. Conclusions. There exists a carrier-mediated transport system for monocarboxylic acids in oral mucosal epithelial cells.     

Demonstration of 3,4-dihydroxy(14C)benzoic acid and (14C)vanillic acid after administration of (14C)noradrenaline in the rat

Rats were injected with 40 μCi (±)-[1-³H]noradrenaline and 10 μCi ±-[1-¹⁴C]noradrenaline. Three fractions with a decreased ³H:¹⁴C ratio were isolated from the urine by a combined alumina adsorption-ethyl acetate extraction procedure. Two of the fractions were identified as [¹⁴C]vanillic acid and 3,4-dihydroxy[¹⁴C]benzoic acid, respectively. Vanillic acid represented between 1.5 and 3.0% of the total [¹⁴C]activity excreted within 24 h and the contribution of dihydroxybenzoic acid was 0·2-0·5%. The third fraction with a decreased ³H: ¹⁴C ratio has not been identified and represented about 2% of the total [¹⁴C]activity excreted within 24 h. After monoamine oxidase blockade with 100 mg/kg of iproniazid, the excretion of vanillic acid, 3,4-dihydroxybenzoic acid and the unknown fraction was greatly diminished. The probability that these three substances represent those metabolites arising simultaneously with the formation of tritium water from (±)-[1-³H]noradrenaline is discussed.     

Some factors regulating [1-14C] acetate incorporation into aflatoxins by spheroplasts and spheroplast lysates from Aspergillus parasiticus

Factors regulating aflatoxin biosynthesis were studied in spheroplasts and lysates of Aspergillus parasiticusNRRL 3240. Over a 2 hr period, ribose, sucrose and maltose stimulated the incorporation of [1-¹⁴C] acetate into aflatoxins. Of a number of inorganic salts studied, zinc sulphate, magnesium sulphate, cobaltous chloride, cadmium acetate and strontium chloride stimulated the incorporation of labeled acetate into aflatoxins. Vanadyl sulphate and ferrous sulphate inhibited de novo aflatoxin formation in spheroplast lysates. Strontium chloride at concentrations of 0·01–0·5 mM stimulated [1-¹⁴C] acetate incorporation. Silver nitrate, copper sulphate and chlorides of manganese, calcium and barium markedly stimulated the incorporation of radioactivity into aflatoxins. An inverse correlation between the incorporation of [1-¹⁴C] acetate into aflatoxins and lipids was noted. In lysates, addition of pyruvic acid markedly increased de novo aflatoxin synthesis. Acetoacetate and bicarbonate enhanced incorporation of radioactivity at lower concentrations. Citric acid at 1–10 mM concentrations was stimulatory to incorporation of label into aflatoxins while malonic acid was inhibitory. In lysates, EDTA enhanced acetate incorporation into aflatoxins and inhibited incorporation of label in intact spheroplasts. This effect was only partially reversed by the addition of ZnSO₄ or MgSO₄.     

14C-棉酚:棉花幼苗生物合成的最佳条件(英文)

将[1-¹⁴C]-乙酸钠加入棉花(Gossypium hirsutum c.v.ST-506)幼苗的培养液中,经四天的培育,得到最高放射活性的棉酚。实验中亦显示体外加乙酸钠具有激发棉酚生物合成的特性。每两百株棉花幼苗加入6 mg乙酸钠可得最高量的棉酚。     

Membrane-associated proteins of adriamycin sensitive and resistant murine leukemic P388 cells

We have isolated an 84-fold adriamycin resistant subline, P388/R84, from mouse leukemia P388 cells by serial cultivation in methylcellulose in the presence of increasing drug concentrations. Electrophoresis of detergent soluble fractions of radiolabeled sensitive and resistant cells suggested marked alterations in the protein fractions of 160, 100, 60, 45, and 30 kd. In resistant clones labeled with ¹²⁵1 an increase in 160 and 100 kd proteins was accompanied by concomitant reduction in the 60, 45, and 30 kd proteins. In ³⁵S methionine-labeled resistant cells, similar increases in the 160 and 100 kd components were observed but in contrast to ¹²⁵I-labeled cells the 30 kd component was also higher. Alterations in surface proteins were confirmed in experiments where the cell extracts were adsorbed to concanavalin A polymers and extracted with 0.26 M methyl-α-D-mannopyranoside. Our data confirm earlier reported observations on cell-surface protein changes in cells resistant to anthracyclines and alkaloids.     

Alkylation of RNA by vinyl chloride metabolites in vitro and in vivo: Formation of 1-N6-etheno-adenosine

Rat liver microsomes were incubated with NADPH, 1,2-[¹⁴C]vinyl chloride and poly-adenosine. The latter was reisolated from the incubations and hydrolyzed. The radioactivity, originating from [¹⁴C]vinyl chloride, which was irreversibly attached to the poly-adenosine was confined to 1-N⁶-etheno-adenosine (3β-ribofuranosyl-imidazo[2,1,i] purine). When rats were exposed to 1,2-[¹⁴C]vinyl chloride, part of the radioactivity was incorporated into RNA of liver. This radioactive labelling exhibited a first maximum, 14 h, and a second maximum, 72 h after ending the exposure. Analysis of hydrolysate of liver RNA showed that all natural nucleosides of RNA were labelled. Besides, small amounts of radioactivity could be detected which were confined to 1-N⁶-etheno-adenosine. The experiments support the theory that vinyl chloride metabolites react with adenosine moieties of nucleic acid under formation of 1-N⁶-etheno-adenosine. Furthermore, the results show that measurement of incorporation of radioactivity into nucleic acids after exposure of animals to radioactive vinyl chloride is not applicable as a means of determining the alkylating potency of vinyl chloride metabolites towards nucleic acids in vivo.