Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Effect of Chlorpromazine on Active Transport of Amino Acids in Tetrahymena*

SYNOPSIS. Low concentrations of chlorpromazine (～0.01 mM) inhibit growth and nucleic acid synthesis in the ciliate Tetrahymena pyriformis. Brief exposure of the cells to, e.g. 0.018 mM chlorpromazine, had very little effect on ¹⁴CO₂ production or on label incorporation into glycogen from [1-¹⁴C]glucetate, [6–14C]glucose, or [1-¹⁴C]leucine, but 17-h exposure of stationary phase cultures to this drug caused marked alterations in metabolism, including an almost complete loss of ability to decarboxylate L-[1-¹⁴C]leucine and L-[1-¹⁴C]tyrosine. It was shown that loss of ability to decarboxylate these amino acids results from loss of ability to transport them.     

Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria

The initial activation reactions of anaerobic oxidation of the aromatic hydrocarbons toluene and ethylbenzene were investigated in cell extracts of a toluene-degrading, sulfate-reducing bacterium, Desulfobacula toluolica, and in cell extracts of strain EbN1, a denitrifying bacterium capable of degrading toluene and ethylbenzene. Extracts of toluene-grown cells of both species catalysed the addition of fumarate to the methyl group of [phenyl-¹⁴C]-toluene and formed [¹⁴C]-labeled benzylsuccinate. Extracts of ethylbenzene-grown cells of strain EbN1 did not catalyse this reaction, but catalysed the formation of 1-phenylethanol and acetophenone from [methylene-¹⁴C]-ethylbenzene. Toluene-grown cells of D. toluolica and strain EbN1 synthesised highly induced polypeptides corresponding to the large subunits of benzylsuccinate synthase from Thauera aromatica. These polypeptides were absent in strain EbN1 after growth on ethylbenzene, although a number of different polypeptides were highly induced. Thus, formation of benzylsuccinate from toluene and fumarate appears to be the general initiating step in anaerobic toluene degradation by bacteria affiliated with the phylogenetically distinct β-subclass (strain EbN1 and T. aromatica) and δ-subclass (D. toluolica) of the Proteobacteria. Anaerobic ethylbenzene oxidation proceeds via a different pathway involving a two-step oxidation of the methylene group to an alcohol and an oxo group; these steps are most probably followed by a biotin-independent carboxylation reaction and thiolytic cleavage. Received: 16 March 1998 / Accepted: 27 June 1998     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

Incorporation of Label from Acetate and Laurate into the Mannan of Leishmania donovani via the Glyoxylate Cycle

Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-¹⁴C]acetate and a 2.4-fold increase from [1-¹⁴C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-¹⁴C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.     

Formation of terpenoid products in Ginkgo biloba L. cultivated cells

Ginkgolides are diterpenes arising from the terpenoid precursor: geranylgeranyl pyrophosphate (GGPP). Incorporation of [1-¹⁴C] isopentenylpyrophosphate ([1-¹⁴C]IPP) into GGPP was monitored throughout the cultivation cycle of G. biloba L. cultivated cells. Because incorporation of [1-¹⁴C]IPP into GGPP had never been monitored in G. biloba, in either the whole plant or cultivated cell system, modifications to existing protocols were necessary. Modifications consisted of extracting the cells with an extraction buffer supplemented with Triton-X-100. Farnesylpyrophosphate (FPP) was the major product formed. The amount of GGPP detected was about one tenth that of FPP.Abbreviations CHAPS 3-[(3-cholamidopropyl) dimethyl ammonio]-1-propane-sulphonate - DTT [1-4 dithiothreitol] - FPP farnesylpyrophosphate - GGPP geranylgeranylpyrophosphate - IPP [1-¹⁴C] isopentenylpyrophosphate - PVPP polyvinylpolypyrrolidone - Tris Tris(hydroxymethyl)aminomethane     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

Activity of the pentose phosphate pathway during lignification

Summary We did this work to see if there is a correlation between lignin synthesis and the activity of the pentose phosphate pathway. Excision of the third internode of the stem of Coleus blumei Benth. followed by incubation on sucrose and indoleacetic acid led to extensive formation of tracheids. During this lignification we determined the activities of glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphate aldolase, and the extent to which [1-¹⁴C]-,[3,4-¹⁴C]-, and [6-¹⁴C]glucose labelled CO₂ and the major cellular components. The results indicate that the pentose phosphate pathway was active during lignification, and that the activity of this pathway relative to glycolysis increased at the onset of lignification. Explants of storage tissue of Helianthus tuberosus L. were cultured under conditions which caused extensive lignification. ¹⁴CO₂ production from [1-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose indicated activity of the pentose phosphate pathway during tracheid formation. We suggest that lignification is accompanied by appreciable activity of the pentose phosphate pathway and that this could provide the reducing power for lignin synthesis.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - IAA indoleacetic acid     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1-¹⁴C]acetate, [2-¹⁴C]acetate, [4-¹⁴C]acetoacetate, [1-¹⁴C]butyrate, [1, 5-¹⁴C]citrate, [2-¹⁴C]glucose, [5-¹⁴C]glutamate, 3-hydroxy[3-¹⁴C]butyrate, [2-¹⁴C]lactate, [U-¹⁴C]leucine, [2-¹⁴C]pyruvate and [³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of [2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from [5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from [1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from [2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from [1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of [1-¹⁴C]acetate, [2-¹⁴C]acetate, [1-¹⁴C]butyrate, [1,5-¹⁴C]citrate, [³H]acetylaspartate, [U-¹⁴C]leucine, and also after [2-¹⁴C]pyruvate and [4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected [2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from [1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of [1-¹⁴C]acetate than after [2-¹⁴C]pyruvate. The results indicate that the [1-¹⁴C]acetyl-CoA arising from [1-¹⁴C]acetate does not enter the same pool as the [1-¹⁴C]acetyl-CoA arising from [2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of [1-¹⁴C]acetate was different from that after [1, 5-¹⁴C]citrate. This suggests that [1-¹⁴C]acetate and [1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.     

A RELATIONSHIP OF N-ACETYLASPARTATE BIOSYNTHESIS TO NEURONAL PROTEIN SYNTHESIS12

A detailed time study of the incorporation of label from sodium-[1-¹⁴C]acetate, [1-¹⁴C]ethanol, and [2-¹⁴C]glucose into the aspartyl moiety of N-acetylaspartic acid (NAA) was conducted. As expected the specific activity of aspartate increased rapidly with time and peaked within 15-20 min after which it fell sharply; but significantly, that of the aspartyl moiety of NAA rose very slowly even after the specific activity of aspartate had fallen to less than 1 per cent of the peak values. A rat brain microsomal free supernatant preparation was shown enzymatically to incorporate label from sodium-[1-¹⁴C]acetate into the t-RNA fraction from which was isolated N-[1-¹⁴C]acetylaspartic acid. From these observations we were inclined to speculate that NAA-t-RNA may serve as an initiator of neuronal protein synthesis.     

Stimulatory effect of gonadotropins on the synthesis of adenosine 3′ :5′-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells

A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase. The isolated cells were capable of converting [8-¹⁴C]adenine to cyclic [¹⁴C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin. The accumulation of progesterone was also measured in these cells by radioimmunoassay. In vitro addition of luteinizing hormone stimulated the accumulation of radioimmunoassayable progesterone. The conversion of [8-¹⁴C]adenine to cyclic [¹⁴C]AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium. Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h. The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic [¹⁴C]AMP formation from [8-¹⁴C]-adenine. Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation, with no effect on cyclic AMP formation. The results suggest that the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Metabolic fate of guanosine in higher plants

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8-¹⁴C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8-¹⁴C]guanine. The rate of uptake of [8-¹⁴C]guanosine increased with the age of the culture. Pulse-chase experiments with [8-¹⁴C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8-¹⁴C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of ¹⁴CO₂ as the final product. The rate of salvage of [8-¹⁴C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8-¹⁴C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during ‘chase’ periods. In segments of young leaves of Camellia sinensis, [8-¹⁴C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO₂ during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by ‘salvage’ reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.     

METABOLISM OF MALONIC ACID IN RAT BRAIN AFTER INTRACEREBRAL INJECTION

Labeled malonic acid ([1-¹⁴C] and [2-¹⁴C]) was injected into the left cerebral hemisphere of anesthetized adult rats in order to determine the metabolic fate of this dicarboxylic acid in central nervous tissue. The animals were allowed to survive for 2, 5, 10. 15 or 30min. Blood was sampled from the torcular during the experimental period and labeled metabolites were extracted from the brain after intracardiac perfusion. There was a very rapid efflux of unreacted malonate in the cerebral venous blood. Labeled CO₂ was recovered from the venous blood and the respired air after the injection of [1-¹⁴C]malonate but not after [2-¹⁴C]malonate. The tissue extracts prepared from the brain showed only minimal labeling of fatty acids and sterols. Much higher radioactivity was present in glutamate, glutamine, aspartate, and GABA. The relative specific activities (RSA) of glutamine never rose above 1.00. Aspartate was labeled very rapidly and revealed evidence of ¹⁴CO₂ fixation in addition to labeling through the Krebs cycle. GABA revealed higher RSA after [1-¹⁴C]malonate than after [2-¹⁴C]malonate. Sequential degradations of glutamate and aspartate proved that labeling of these amino acids occurred from [1-¹⁴C] acetyl-CoA and [2-¹⁴C] acetyl-CoA, respectively, via the Krebs cycle. Malonate activation and malonyl-CoA decarboxylation in vivo were similar to experiments with isolated mitochondria. However, labeled malonate was not incorporated into the amino acids of free mitochondria. The results were compared to data obtained after intracerebral injection of [1-¹⁴C]acetate and [2-¹⁴C]acetate.     

Binding site of novel 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5- triazine herbicides in the D1 protein of Photosystem II

A series of replacement experiments of [¹⁴C]-triazines, [¹⁴C]-atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [¹⁴C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [¹⁴C]-Atrazine and [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-¹⁴C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [¹⁴C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Hydrocortisone selectively inhibits IgE-dependent arachidonic acid release from rat peritoneal mast cells

Purified rat mast cells were used to study the effects of anti-inflammatory steroids on the release of [1-¹⁴C]-arachidonic acid ([1-¹⁴C]AA) and metabolites. Mast cells were incubated overnight with glucocorticoids, [1-¹⁴C]AA incorporated into cellular phospholipids and the release of [1-¹⁴C]AA, and metabolites determined using a variety of secretagogues. Release of [1-¹⁴C]AA and metabolites by concanavalin A, the antigen ovalbumin and anti-immunoglobulin in E antibody was markedly reduced by glucocorticoid treatment. Neither the total incorporation of [1-¹⁴C]AA nor the distribution into phospholipids was altered by hydrocortisone pretreatment. Glucocorticoid pretreatment did not alter [1-¹⁴C]AA release stimulated by somatostatin, compound 48/80, or the calcium ionophore, A23187. These data indicate that antiinflammatory steroids selectively inhibit immunoglobulin dependent release of arachidonic acid from rat mast cells. These findings question the role of lipomodulin and macrocortin as general phospholipase inhibitors and suggest that they may be restricted to immunoglobulin stimuli.     

Relationship between the pentose-phosphate pathway and the de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cultures

This study focuses on the activity of the pentose-phosphate pathway and its relationship to de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cell cultures derived from 1-week old rat brain. The proportion of glucose that was metabolized along the pentose-phosphate pathway was estimated by measuring ¹⁴CO₂ production from [1-¹⁴C]-, [2-¹⁴C]- and [6-¹⁴C]glucose, the utilization of glucose and the production of lactate. Incorporation of ¹⁴C from [¹⁴C]glucose and from [3-¹⁴C]acetoacetate into lipids was analysed. The pentose- phosphate pathway produced much more CO₂ from glucose than the Krebs cycle, although it accounted for only a small part of the consumption of glucose (< 3%). The higher ¹⁴CO₂ production from [2-¹⁴C]glucose than from [6-¹⁴C]glucose indicated that recycling of the products of the pentose-phosphate pathway takes place in these cells.Gradual inhibition of the pathway with increasing concentrations of 6-aminonicotinamide resulted in a parallel inhibition of the conversion of acetoacetate and of glucose into fatty acids and into cholesterol. Glycolysis was also strongly inhibited in the presence of 6-aminonicotinamide whereas the activity of the Krebs cycle was not affected.These results suggest that de novo synthesis of fatty acids and cholesterol by oligodendrocytes of neonatal rats is closely geared to the activity of the pentose-phosphate pathway in these cells.