The synthetic potential of immobilised cells of Capsicum frutescens Mill cv. annuum

Cells of Capsicum frutescens Mill. cv. annuum, immobilised in reticulate polyurethane foam, produced higher yields of capsaicin, the pungent principle of Chilli pepper fruits, than did freely-suspended cells, when batch-cultured in a medium conducive to culture growth. In the absence of specific precursors to capsaicin, immobilised cells produced between two and three orders of magnitude higher yields than did suspended cells over 5-d or 10-d culture periods (typically up to 4 or 5 mg capsaicin g^-1 dry weight l^-1 medium compared with up to 30 g g^-1l^-1, respectively). These results were reflected by an increased rate and extent of incorporation of L-[U-¹⁴C]phenylalanine into capsaicin in immobilised as compared with freely-suspended cells, and evidence is presented for an inverse relationship between incorporation of [¹⁴C]phenylalanine into protein and capsaicin. The accumulation of capsaicin can be experimentally manipulated and increased by supplementing the medium with precursors of capsaicin such as phenylalanine and isocapric acid and by reducing the growth rate of immobilised cells by omitting growth regulators from the medium. The importance of these observations is discussed.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine - TLC thin-layer chromatography     

The influence of intracellular pools of phenylalanine derivatives upon the synthesis of capsaicin by immobilized cell cultures of the chilli pepper,Capsicum frutescens

In an attempt to determine the potential factors controlling the biosynthesis of the secondary metabolite capsaicin by immobilized cell cultures of the chilli pepper, Capsicum frutescens Mill, labelling techniques using the radioactive precursor [¹⁴C]phenylalanine have been employed. Following preincubation treatments with either capsaicin (the end-product of the pathway) or sinapic acid, [¹⁴C]phenylalanine was applied and the movement of the label through the pathway and its eventual fate was followed. Results have shown that capsaicin, through a feedback-inhibition mechanism, negatively influences its own synthesis. Furthermore, capsaicin synthesis in these cells is not controlled via the activity of the enzymes phenylalanine ammonia-lyase and cinnamate 4-hydroxylase which may determine the rate of entry of metabolites into the phenylpropanoid pathway. The importance of other sinks for phenylalanine derivatives, which may compete for capsaicin precursors, has also been investigated. Surprisingly, protein proved to be only a relatively minor sink for phenylalanine with the great majority of the label rapidly ending up in covalently bound phenolics in the cell wall. Attempts to prevent this by applying sinapic acid were only partially successful. The importance of these results in relation to the possible control mechanisms which operate to control secondary metabolite synthesis in vitro is discussed.     

Variation in the growth and biosynthetic activity of cloned cell cultures of Capsicum frutescens and their response to an exogenously supplied elicitor

Twenty clones established from single cells of a suspension culture of Capsicum frutescens were maintained as callus and in suspension over a sixteen week culture period. These clones exhibited marked differences in growth, chlorophyll and chloroform-soluble phenolic content which became more apparent with increasing time in culture. Clones in suspension exhibited a more rapid change in morphology and biosynthetic activity than those cultured as callus. Elicitation increased PAL activity, reduced the incorporation of L-[U-¹⁴C] phenylalanine into the chloroform-soluble fraction of the culture medium and increased incorporation into the methanol-soluble fraction of the cells in ten suspension clones. Differences to elicitation were observed among clones; in particular the faster growing isolates incorporated more radioactive label into soluble phenolics that remain in the cells than those that are released into the medium. The implications of these results are discussed.Abbreviations SH Schenk & Hildebrandt - PAL phenylalanine ammonia-lyase - RGR relative growth rate - TCC total chlorophyll content - HPLC high performance liquid chromatography     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili

Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g^?1 f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g^?1 f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g^?1 f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g^?1 f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g^?1 f.wt on day 20 and 1,315.3 ± 10 μg g^?1 f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.     

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.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Hexitols as major intermediates of glucose assimilation by mycelium of Puccinia graminis

Mycelium of Puccinia graminis was grown for 4 d on 200 mM D-[U-¹⁴C]glucose followed by a cold chase for 30 h. Analysis of cellular metabolites during the chase indicated significant turnover only in carbohydrates soluble in 80% (w/v) ethanol. A kinetic analysis of the depletion of [¹⁴C] in pools of free sugars and sugar alcohols indicated that the trehalose pools and a small proportion (12–16%) of the mannitol and glucitol pools did not turn over, whilst pools of glucose, fructose, and the remainder of the hexitols became totally,depleted of label during the chase. Because the [¹⁴C] was totally lost from the pools of glucose and fructose prior to the hexitols, it was deduced that both of these hexoses were precursors of the hexitols. Estimation of the carbon fluxes through pools indicated that 52, 36 and 16% of the carbon from glucose was assimilated via glucitol, fructose and mannitol respectively, demonstrating that glucitol could not have originated from fructose as sole precursor. After offering D-[U-¹⁴C]glucitol, [¹⁴C] was assimilated into trehalose phosphate, glucans and amino acids, but not into free glucose or fructose. These data indicate that hexitols are quantitatively important intermediates during the assimilation of glucose by Puccinia graminis.     

An investigation of acid-soluble nuclear proteins of human leucocytes in relation to fraction RP2-L, a component of neoplastic cells

1. The claim that tumour cells contain a specific nuclear protein was investigated. The presence of this component was confirmed in Walker tumour cells by the chromatography on CM-cellulose of nuclear proteins labelled with [¹⁴C]lysine. This protein was studied further in a number of human leucocyte cells. 2. The labelling of leucocyte nuclear proteins with [¹⁴C]lysine was attempted during incubation and culture in vitro. Incorporation of the label into acid-soluble nuclear proteins was highest in normal lymphocytes cultured with phytohaemagglutinin, followed by chronic-myeloid-leukaemic leucocytes and mixed samples of normal leucocytes incubated in plasma. Little incorporation was seen in similar extracts of chronic-lymphatic or normal leucocytes. 3. Lymphocytes were the only cells that gave nuclear extracts with amino acid analysis similar to that of unfractionated histones. 4. Little of the [¹⁴C]lysine in nuclear extracts of incubated leucocytes proved to be of chromosomal origin. No evidence was found of an RP2-L component in the highly labelled nuclear extracts of phytohaemagglutinin-treated lymphocytes until after 6 days of culture with [¹⁴C]lysine. This component was soluble in saline. 5. Evidence is presented that fraction RP2-L is a non-histone protein constituent of cell nuclei whose labelling with [¹⁴C]lysine may be dependent on the metabolic state of the cell. Thus this component is not specific to the neoplastic state.     

Mechanism of irreversible inactivation of phenylalanine-4- and tryptophan-5-hydroxylases by [4-36Cl, 2-14C]p-chlorophenylalanine: A revision

Intraperitoneal injection of [4-³⁶Cl, 2-¹⁴C]p-chlorophenylalanine (pCPA) (300 mg/kg) in rats revealed absence of chlorine in pure hepatic phenylalanine hydroxyase, while the carbon label appeared as 1–4 moles/mole of [¹⁴C]tyrosine in the inactivated phenylalanine and cerebral tryptophan-5-hydroxylase. Crystalline muscle aldolase and tyrosine hydroxylase also revealed the presence of [2-¹⁴C]tyrosine from [2-¹⁴C]pCPA without inactivating these enzymes. Injection of L-[(U)-¹⁴C] tyrosine led to its incorporation into the above enzymes, but to a different degree without altering the enzyme activity. Repeated injections ofp-chlorophenylacetic acid had no effect on phenylalanine or tryptophan-hydroxylase. Administration of pCPA did not change the levels of cerebral biopterins. Reexamination of the effect of cycloheximide on reversing enzymic inactivation by pCPA failed to confirm our earlier observation.     

Effects of cycloheximide,d-threo-chloramphenicol,erythromycin and actinomycin D on De-novo synthesis of cytoplasmic and mitochondrial proteins in the cotyledons of germinating pea seeds

Summary Inhibitors of, and radioactive substrates for, protein synthesis were introduced into germinating pea (Pisum sativum L.) seeds, and protein synthesis was allowed to proceed in vivo. Subsequent analyses of subcellular fractions showed the following: Cycloheximide strongly inhibited the incorporation of [¹⁴C]leucine into both mitochondrial and cytoplasmic proteins. d-Threo-chloramphenicol and erythromycin did not affect cytoplasmic protein synthesis, but partially inhibited mitochondrial protein synthesis. These results suggest that most of the new mitochondrial proteins were originally synthesized in the cytoplasm. Actinomycin D did not appreciably affect the initial incorporation of [¹⁴C]leucine into either mitochondrial or cytoplasmic proteins, suggesting that information (mRNA) concerning the initially synthesized proteins may be present in the quiescent seeds. The lack of appreciable incorporation of [³H]thymidine into mitochondrial DNA supported our previons report that mitochondria may not be synthesized de novo in pea cotyledons.     

Biosynthesis of thyroglobulin. Incorporation of [1-14C]galactose, [1-14C]mannose and [4,5-3H2]leucine into soluble proteins by rat thyroids in vitro

1. Rat thyroid lobes were incubated for various periods of time in Krebs–Ringer bicarbonate containing [³H]leucine and either [1-¹⁴C]galactose or [1-¹⁴C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrose-gradient centrifugation. 2. The time-course of incorporation of label from [¹⁴C]-mannose into soluble thyroid proteins was parallel to that observed for [³H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17–18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3–8s). Radioactivity from double-labelled fractions 12s and 3–8s was transferred to protein 17–18s during subsequent incubation in an unlabelled medium. 3. In contrast, most of the [¹⁴C]galactose was immediately incorporated into protein 17–18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [³H]leucine and [¹⁴C]mannose into all protein fractions, but had little effect on the incorporation of [¹⁴C]galactose into protein 17–18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17–18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of ghyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17–18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17–18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.     

Effects of hydroxyproline on the growth of excised root segments of Pisum sativum under aseptic conditions

Summary The cis and trans isomers of 4-hydroxy-l-proline stimulated the extension growth of excised 2–4 mm pea root segments during culture. Increase in the uptake and subsequent incorporation of [¹⁴c]leucine into proteins was inhibited by both l-isomers, and so also were changes in chloride uptake capacity and in protein metabolism measured in terms of invertase and peroxidase activities. Changes in [¹⁴C]proline uptake and incorporation, and in respiration, were unaffected. Proline had no effect on changes in extension growth or protein metabolism but did prevent the effects of both hydroxyproline isomers. Azetidine-2-carboxylic acid inhibited extension growth and all the aspects of protein metabolism studied, the effects again being all prevented by proline. It is suggested that hydroxyproline enhances growth by interfering with protein synthesis in the cell walls.     

Evidence for a functional change in the plasma membrane of yeasts associated with the mechanism of arsenate resistance.

In previous reports experimental evidence has been presented indicating a possible relationship between the formation of arseno-phosphoinositides and the active transport of arsenate-phosphate in yeast cells. There is an increment in the amount of inositides in yeast cells adapted to grow in the presence of toxic concentrations of arsenate. These cells exhibit a highly reduced arsenate uptake but maintain their capacity to transport phosphate. Since, in normal (nonadapted) yeast cells, both arsenate and phosphate anions share the same transport system, a study was conducted to obtain further information about the plausible role played by the phosphoinositides in the active transport system of arsenate and their inhibition that allows the cells to grow in the presence of the toxic. Studies on [³²P]orthophosphate and [⁷⁴As]arsenate incorporation into phospholipids in normal and arsenate-adapted yeast show that: The ³²P incorporation into phospholipids is two times larger in normal yeast as compared to arsenateadapted ones. The ³²P labeling was maximum for phosphatidylinositol in normal yeasts while in the arsenate-adapted cells it was maximum for phosphatidylcholine. This incorporation was largely inhibited by arsenate in normal yeasts and minimal in the arsenate-adapted ones. Cell fractionation shows that the maximum incorporation of [³²P]orthophosphate resides in the microsomal fraction, while the incorporation of [⁷⁴As]arsenate resides mainly in the cell envelope fraction which incorporates 86% of the ⁷⁴As label. Phosphate is capable of inhibiting the ⁷⁴As-inositide complex formation and destroying the previously formed one. Yeast cells prelabeled with [2C-³H]myoinositol showed a reduced turnover rate of phosphoinositides even when transporting nontoxic amounts of arsenate. The involvement of the inositides as a regulatory mechanism in the phosphate-arsenate active transport system in yeast cells is discussed.     

Organ culture of adult rat colonic mucosa on fibrin foam

Summary A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37°C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum,l-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmosphere for culture was 95% O₂ and 5% CO₂. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [³H]thymidine and [³H]leucine into DNA and protein, [¹⁴C]glucosamine and [³H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O₂ retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland. This work was supported by the National Cancer Institute Contract N01-CP-75953 and in part by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, under Contract N01-CO-65341 with the International Union Against Cancer.     

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine -Nmethyltransferase and -N-methylornithine decafboxylase were undetectable, indicating that -N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-¹⁴C]ornithine, l-[U-¹⁴C]arginine, [U-¹⁴C]agmaine and [1,4-¹⁴C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl--difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl--difluoromethylornithine. Together with the demonstration that label was incorporated from [U-¹⁴C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-¹⁴C] ornithine and l-[U-¹⁴C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.Abbreviations DFMA dl--difluoromethylarginine - DFMO dl--difluoromethylornithine We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.     

Incorporation of labeled small molecules into rubratoxin

A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28° C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1¹⁴C]acetate, [1,5¹⁴C]citrate, [2¹⁴C]malonate, [1¹⁴C]glucose, [U¹⁴C]glucose or [1¹⁴C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1¹⁴C]acetate and [2¹⁴C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.Abbreviations GMS glucose-mineral salts - RCM replacement culture medium - TCA tricarboxylic acid - PEP phosphoenolpyruvate - RIC relative isotopic content - PI percent incorporation     

ELEVATED PLASMA PHENYLALANINE CONCENTRATION AND LYSINE INCORPORATION INTO RIBOSOMAL PROTEIN OF DEVELOPING BRAIN

Hyperphenylalaninemia was induced in 7-day-old rabbits over a 6-hr period by intraperitoneal injection of phenylalanine. l -[U-¹⁴C]Lysine was injected intraperitoneally into these rabbits and into a control group. The rate of incorporation of l -[U-¹⁴C]lysme into brain ribosomal protein was decreased during a 5-hr period in the presence of elevated plasma phenylalanine concentrations. Lysine transport from the peritoneum to the plasma was unaffected by the high plasma phenylalanine concentrations.