L-3-(3,4-Dihydroxyphenyl)alanine (<Emphasis Type="SmallCaps">L</Emphasis>-DOPA), an allelochemical exuded from velvetbean (<Emphasis Type="Italic">Mucuna pruriens</Emphasis>) roots

We investigated whether or not lettuce growth was inhibited by diffused L-3-(3,4-dihydroxyphenyl)alanine (L-DOPA), an allelochemical exuded from the roots of velvetbean (Mucuna pruriens (L.) DC. var. utilis) cultivars using a modified plant-box bioassay. For all the cultivars and one accession examined L-DOPA diffused from the roots and caused radicle and hypocotyl growth inhibition. A high correlation co-efficient (r = 0.838 to 0.982) was observed between L-DOPA concentration and lettuce seed sowing distance. L-DOPA diffused equally in all directions from roots at 0 mm position (close to root surface) in the plant-box, while the inhibition (%) of lettuce radicle growth gradually decreased with distance from the roots. For all cultivars the concentration of L-DOPA was significantly different at 0 mm position: being highest in cv. preta (167 g/ml) and lowest in cv. jaspeada and cv. ana (13 g/ml). The correlation between lettuce radicle growth inhibition and concentration of diffused L-DOPA was high (r = 0.856 to 0.966) in all cultivars and accession examined. However, the concentration of diffused L-DOPA did not correlate with the fresh weight concentration of L-DOPA measured in roots. The lettuce radicle growth inhibition from mucuna diffused L-DOPA was very similar that induced by synthetic L-DOPA, suggesting that diffused L-DOPA was the allelochemical responsible for growth inhibition.     

Clostridium methylpentosum sp. nov.: a ring-shaped intestinal bacterium that ferments only methylpentoses and pentoses

An intestinal bacterium isolated from a human subject utilized only two methylpentoses (L-rhamnose and L-fucose) and two pentoses (L-lyxose and D-arabinose) as fermentable substrates, among many compounds tested. The isolate was obligately anaerobic and had a distinctive morphology, its cells being rods bent in the shape of rings with the ends slightly overlapping. Single ring-shaped cells and left-handed helical chains of cells were present in cultures. The cells were surrounded by large capsules which appeared as thick, fibrous masses when examined by electron microscopy. Capsules were formed by cells growing in media containing any one of the four fermentable substrates. Terminally located, heat-resistant endospores were formed on plates of an enriched agar medium supplemented with L-rhamnose. End products of L-rhamnose or L-fucose fermentation included acetate, propionate, n-propanol, CO₂, and H₂. The isolate represented a new species of Clostridium for which the name Clostridium methylpentosum (type strain R2. ATCC 43829) is proposed. This organism may participate in intestinal digestive processes by metabolizing rhamnose released via the enzymatic depolymerization of dietary pectin.Abbreviations G+C guanine plus cytosine - OD optical density - TEM transmission electron micrograph     

Modulation of striatal dopamine release in cerebral ischemia byL-arginine

The effect ofL-arginine, the precursor of nitric oxide, on ischemic dopamine release from the striatum was investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (2 h). Dopamine and its metabolites were measured in the striatal extracellular space dialysate after continuous perfusion (2 l/min) of artificial extracellular fluid in the presence or absence of 15 mmol/literL- orD-arginine or 1 mmol/liter nitro-L-arginine.L-Arginine but notD-arginine increased the striatal content of dopamine in pre- and postischemia whereas it lowered the levels of dopamine and 3-methoxytyramine induced by ischemia. In contrast, nitro-L-arginine reduced the preischemic levels of dopamine and 3,4-dihydroxyphenyl-acetic acid, and had no effect on the ischemic release of dopamine. These findings indicate thatL-arginine stereospecifically modified the ischemic release and metabolism of dopamine. The data also suggest that the basal level of nitric oxide is not involved in dopamine release during ischemia but may participate in regulating dopamine release under physiological conditions.Presented in part at the 19th International Joint Conference on Stroke and Cerebral Circulation, San Diego, California, February 17–19, 1994.     

Enzymatic Synthesis and Antioxidant Properties of <Emphasis Type="SmallCaps">L</Emphasis>-Ascorbyl Oleate and <Emphasis Type="SmallCaps">L</Emphasis>-Ascorbyl Linoleate

L-Ascorbyl oleate and L-ascorbyl linoleate were synthesized by an immobilized lipase from Candida antarctica with yields of 38% and 44%, respectively. L-Ascorbyl oleate was stable in sterile culture medium over 12 h at 37 °C but L-ascorbyl linoleate degraded by 17%. Ascorbyl oleate had a better protective effect on human umbilical cord vein endothelial cells treated with H₂O₂ than of L-ascorbic acid-2-phosphate-6-palmitate (Asc2P6P).Revisions requested 21 July 2004/26 August 2004; Revisions received 20 August 2004/27 September 2004     

Formation of L-canavanine in in vitro cultures of Canavalia ensiformis (L.) DC.

In vitro tissue cultures of Canavalia ensiformis (L.) D.C. derived from hypocotyl have been obtained. They were found to accumulate L-canavanine depending on the medium where they were grown. Addition of polyethylenglycol (4%) to the culture medium led to a reduced accumulation of l-canavanine and an increase in the amino acids and the quaternary ammonium compounds contents.     

Direct and decarboxylation-dependent effects of neurotransmitter precursors on firing of isolated monoaminergic neurons

To elucidate mechanisms that underlie the profound physiological effects of the monoamine precursors 5-hydroxy-l-tryptophan (5-HTP) and l-3,4-dihydroxyphenylalanine (l-DOPA), we examined their action on single monoaminergic neurons isolated from the ganglia of the gastropod snail Lymnaea stagnalis. In isolated serotonergic PeA motoneurons, 5-HTP produced excitation. The effect was mimicked by serotonin at 0.5–1 μM, masked by pretreatment with serotonin at higher concentrations, and abolished by the inhibitor of aromatic amino acid decarboxylase (AAAD) m-hydroxybenzylhydrazine (NSD-1015), the inhibitor of the vesicular monoamine transporter reserpine or the serotonin receptor antagonist mianserin. Exposure of the dopaminergic interneurons RPeD1 to l-DOPA caused a biphasic effect composed of a depolarization followed by a hyperpolarization. AAAD inactivation with NSD-1015, as well as the blockade of dopamine receptors with sulpiride, resulted in the enhancement of the excitatory effect, and the abolition of the inhibitory effect. Dopamine caused hyperpolarization and masked the inhibitory phase of l-DOPA action. The results show that precursors affect the rate of firing of isolated monoaminergic neurons and that their effect is completely or partially mediated by the enhanced synthesis of the respective neurotransmitter, followed by extrasynaptic release of the latter and activation of extrasynaptic autoreceptors.     

Leucine and serine induce mecillinam resistance in Escherichia coli.

Summary We have previously shown that resistance to the -lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and ReIA-dependent induction of the stringent response. We show here that our E. coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of lleucine or L-serine (or cysteine, which inactivates the antibiotic). The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium. At least in the case of serine, the resistance is ReIA-dependent. We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.     

A novel mechanism involved in the metabolism of the tartaric acid stereoisomers in Rhodopseudomonas sphaeroides: enzymatic conversion of meso-tartaric acid to D(-)-glyceric acid and CO2

In cell extracts of Rhodopseudomonas sphaeroides grown on meso-tartrate the activities of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and 1.1.1.83, respectively) could be measured spectrophotometrically but not the activity of a meso-tartrate dehydrogenase or dehydratase. However, an enzyme activity was detected manometrically that catalyzed the stoichiometric release of CO₂ from mesotartrate in a molar ratio of 1:1. This reaction required catalytic amounts of NAD and the presence of both divalent (Mn²⁺ or Mg²⁺) and monovalent (NH ₄ ⁺ or K⁺) cations. Purification of the meso-tartrate decarboxylase showed that it was part of the bifunctional L(+)-tartrate dehydrogenase-D(+)-malate dehydrogenase (decarboxylating), which thus possessed a third catalytic function. The homogeneous enzyme catalyzed the stoichiometric conversion of incso-tartaric acid to D(-)-glyceric acid and CO₂. All interfering catalytic activities had been eliminated during the course of enzyme purification.     

Cadaverin ist ein Zwischenprodukt der Biosynthese von Arthrobactin und Ferrioxamin E

Zusammenfassung Cadaverin wird mit einer höheren Ausbeute als Lysin in Arthrobactin von Arthrobacter pascens und Ferrioxamin E von Streptomyces glaucescens eingebaut. Von einem racemischen Lysingemisch kann nur das L-Isomere eingebaut werden. Die Aktivität der L-Lysindecarboxylase wurde in vivo und in vitro gemessen. Das Enzym ist durch Lysin nicht induzierbar und wird durch 5·10^-6 M Fe³⁺ vollständig reprimiert. Bei Klebsiella pneumoniae, dem Produzenten von Aerobactin, konnte nur eine geringfügige Lysindecarboxylase-Aktivität nachgewiesen werden.

---

Cadaverin is an intermediate in the biosynthesis of arthrobactin and ferrioxamin E

Cadaverin was more readily incorporated than lysine into arthrobactin from Arthrobacter pascens and into ferrioxamin E from Streptomyces glaucescens. From a racemic mixture only the L-isomer of lysine is incorporated. The L-lysine decarboxylase activity was measured in vivo and in vitro. The enzyme from Arthrobacter pascens is not inducable by lysine and completely repressed by 5·10^-6 M Fe³⁺. In Klebsiella pneumoniae, the producer of aerobactin, only a very low activity of L-lysine decarboxylase was detected.

     

Electrophoresis of human L-glutamate dehydrogenase: Tissue distribution and preliminary population survey

A method for the starch gel electrophoresis of human L-glutamate dehydrogenase (GLUD) is described, as is the tissue distribution of GLUD detected by this method. Extracts of livers from 200 Whites were analyzed without demonstration of an electrophoretic variant. The molecular size was estimated to be 330,000 and the isoelectric point pH 4.83.This investigation was supported by Public Health Service Grant No. 1 F22 CAO2083-01 of the National Cancer Institute.     

Purification and properties of a phenol oxidase derived from suspension cultures of Mucuna pruriens

From cells of Mucuna pruriens, grown in suspension, a monophenol monooxygenase (EC 1.14.18.1) was purified to homogeneity, as deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme appeared to have a native molecular weight of 90000±5000 dalton, and consisted of two subunits, each of 42000±1000 dalton. High-performance liquid chromatography with electrochemical detection for specific measurement of catecholes, was used to determine separately the tyrosinehydroxylating and catecholase activities of the enzyme. For the enzymatic activities, pH optima of, respectively, 7.5 and 5.5–6.5 were found; the effects of some inhibitors on both activities appeared to be different. Michaelis-Menten characteristics for some mono-and o-dihydroxysubstrates were determined.Abbreviations DEAE diethylaminoethyl - HPLC high-performance liquid chromatography - L-DOPA L-3,4-dihydroxyphenylalanine     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

The pathway ofD-cycloserine biosynthesis inStreptomyces garyphalus

Incubation experiments using washed cells and toluene treated cells ofStreptomyces garyphalus showed that O-acetyl-L-serine and hydroxyurea are intermediates in the biosynthesis ofD-cycloserine. The formation of [¹⁴C]O-ureidoserine from O-acetyl-L-serine and hydroxyurea was demonstrated by incubating an enzyme solution with¹⁴C-labelled substrates. Desalted cell-free extract catalyzed the conversion of O-ureido-D-serine toD-cycloserine in a reaction requiring ATP and Mg²⁺. The results suggested the following pathway forD-cycloserine biosynthesis.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

A Metal Ion as a Cofactor Attenuates Substrate Inhibition in the Enzymatic Production of a High Concentration of <Emphasis Type="SmallCaps">D</Emphasis>-glutamate Using <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">D</Emphasis>-glutamate Amidohydrolase

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co²⁺ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co²⁺ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn²⁺ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.     

Effects of methylammonium and ofL-methionine-DL-sulfoximine on the growth and nitrogen metabolism ofPhaeodactylum tricornutum

Phaeodactylum tricornutum Bohlin grew well withL-methionine-DL-sulfoximine (MSX) as sole nitrogen source. Such growth helps to explain the lack of effect of MSX on ammonium assimilation by this organism. Methylammonium inhibited growth with nitrate or MSX as sole nitrogen source but not growth on ammonium. Methylammonium could not be metabolised byP. tricornutum but was accumulated in the cells, the concentration factor sometimes approaching 25,000. Ammonium addition, but not that of MSX or nitrate, displaced methylammonium from the cells and this displacement was followed by resumption of growth. Both methylammonium and ammonium inhibited the uptake of nitrate and nitrite by the cells but inhibition by methylammonium, in comparison with that by ammonium, required a higher concentration and a longer time to develop. Inhibition by methylammonium is shown to be associated with its accumulation by the cells. Methylammonium also prevented the disappearance of nitrate from the interior of the cells (presumably by nitrate assimilation) whereas ammonium did not. It is concluded that methylammonium and ammonium differ in the ways in which they inhibit nitrate metabolism inP. tricornutum.Abbreviation MSX L-methionine-DL-sulfoximine     

Chiral Selection in Oligoadenylate Formation in the Presence of a Metal ion Catalyst or Poly(U) Template

The lead ion-catalyzed oligomerization of 5′-phosphorimidazolides of D-, L- or racemic DL-adenosine (D-ImpA, L-ImpA and DL-ImpA) gave oligoadenylates up to a pentamer. The oligomers resulting from racemic ImpA were comparable in yields and length to those from chiral D- or L-ImpA. A complex mixture of homochiral and heterochiral oligomers was formed in the reaction from racemic ImpA. Total dimer product from racemic ImpA by the lead ion catalyst showed homochiral selectivity. The reaction catalyzed by uranyl ion yielded oligoadenylates up to 15mer from chiral D- or L-ImpA in over 95% yield. A complex mixture of isomeric oligoadenylates was formed from racemic DL-ImpA in the presence of uranyl ion catalyst in comparable yields to those from D- or L-ImpA. The analysis of the dimer product from DL-ImpA showed that the homochiral 2′ –5′ linked dimer was selectively formed. D-ImpA polymerized effectively on a poly(U) template, which is exclusively composed of D-uridine, yielding oligoadenylates up to a pentamer. In contrast, L-ImpA or racemic DL-ImpA polymerized far less efficiently on the poly(U) template, demonstrating that chiral selection takes place in the poly(U) template-directed oligoadenylate formation.     

Variation in alpha-L-fucosidase properties among 28 inbred mouse strains: Six strains have high enzyme activity and heat-stabile enzyme with a variant pH-activity curve; twenty-two strains have low activity and heat-labile enzyme

Alpha-L-fucosidase in tissues of 28 inbred mouse strains varied with respect to three properties: high or low heat stability, a pH-activity curve with high or low relative activity at pH 2.8, and high or low activity. Alpha-L-fucosidase from six strains (A/J, BDP/J, LP/J, P/J, SEA/GNJ, and 129/J) had high heat stability, high pH 2.8 relative activity, and high activity, whereas the other 22 strains all had low heat stability, low pH 2.8 relative activity, and low activity. The heat-stability difference was seen in all organs tested (brain, liver, kidney, spleen, heart, skeletal muscle, lung, and testis) for two heat-stabile strains (P/J and 129/J) and four heat-labile strains (C57 BL/6J, C3H/HeJ, DBA/2J, and BALB/cJ) studied in detail. The findings suggested that two structural variants of alpha-L-fucosidase, probably genetically determined, exist in these 28 inbred mouse strains, although the presence of linkage disequilibrium between alleles of tightly linked structural and regulatory genes could not be excluded.This work was supported by grants from the National Institutes of Health (NS-15281 and NS-11766), the Muscular Dystrophy Association (H. Houston Merritt Clinical Center for Muscular Dystrophy and Related Diseases), the March of Dimes Birth Defects Foundation, and a generous gift from the Alexander Rapaport Foundation.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.