The cytosolic pathway of l-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase

Saccharomyces cerevisiae accumulates l-malic acid but only minute amounts of fumaric acid. A ¹³C-nuclear magnetic resonance study following the label from glucose to l-malic acid indicates that the l-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to l-malic acid is responsible for the l-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to l-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards l-malic acid; the K _m for l-malic acid is very high indicating that l-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more l-malic acid). (c) According to ¹³C NMR studies there is no interconversion of cytosolic l-malic and fumaric acids.     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Transport of malic acid in Leuconostoc oenos strains defective in malolactic fermentation: a model to evaluate the kinetic parameters

Two Leuconostoc oenos mutant strains unable to metabolize malic acid were differentiated by [U-¹⁴C]-labelled L-malate transport assays into a malolactic-enzyme-deficient mutant and a malate-transport-defective mutant. A mathematical analysis of the data from L-malic acid uptake at three pH values (5.2, 4.5, and 3.2) in the malolactic-enzyme-deficient strains suggest two simultaneous uptake mechanisms, presumably a carrier-mediated transport and a passive diffusion for the anionic and the undissociated forms of the acid, respectively. The apparent affinity constant (K _m t) and the maximal rate (V _m t) values for L-malate active transport were, 12 mM and 43 mol L-malate·mg^–1·s^–1, respectively. Active transport was constitutive and strongly inhibited by protonophores and by ATPase inhibitors. L-Lactic acid appeared to inhibit L-malic acid transport, suggesting an L-lactate/L-malate exchange. At pH values of 4.5 or above, the passive diffusion of L-malic acid was negligible. However, at pH 3.2, the mean pH of wine, the permeability of the cells to the undissociated acid by simple diffusion could represent more than 50% of total L-malic acid uptake, with a diffusion constant (K _D) of 0.1 s^–1. Correspondence to: C. Divies     

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.     

d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q₁, thus distinguishing it from d-amino acid oxidase. The apparent K _m and V _max values were 40.2 mM and 25.0 μmol min⁻¹ mg⁻¹, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min⁻¹ mg⁻¹, respectively, for reduction of Q₁. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.     

Substrataufnahme und Phosphatstoffwechsel bei Ankistrodesmus braunii

Summary The findings of Jacobi (1959) that glycolate enhances the uptake and the incorporation of ³²P-labelled orthophosphate were re-investigated. In contrast to the findings of Jacobi we found that glycolic acid has no effect at concentrations between 10^-3 and 10^-7M. The effect reported by Jacobi was seen only when instead of glycolic acid Na-glycolate was used in the experiments, as it was done by Jacobi. Further experiments showed that the enhancement of ³²P-incorporation is due only to the Na⁺-ions and not to glycolic acid. In addition it was found that up to pH 4,3 no glycolic acid is resorbed from the medium by cells of Ankistrodesmus braunii.     

l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Neuropharmacological analysis of synaptic transmission in the Lorenzinian ampulla of the skate Raja clavata

Summary Dissected ampullae of Lorenzini of the skate (Raja clavata) were studied with the aim of determining the synaptic transmitter between electroreceptor cell and afferent fibre. Resting activity and stimulus-evoked activity in response to electrical pulses were recorded in single afferent units at constant perfusion with normal and test solutions containing different putative neurotransmitters. Presynaptic transmitter release was blocked by Mg²⁺ (up to 50 mM) to investigate the effects of the test substances upon the postsynaptic membrane. l-Glutamate (l-GLU) and l-aspartate (l-ASP), both at concentrations between 10^-7 and 10^-3 M, enlarged strongly resting and stimulus-evoked discharge frequency in the afferent fibre. If transmission was blocked by high Mg²⁺, resting discharge frequency could be restored by l-GLU or l-ASP. The glutamate agonists quisqualate (10^-8–10⁵ M) and N-methyl-D-aspartate (10^-5–10^-3 M) enlarged spontaneous activity in the afferent fiber. The same was found for kainic acid (10^-9–10^-5 M). Taurine at concentrations between 10^-5 and 10^-3 M caused a concentration-dependent decrease in afferent activity. The same was found for gammaaminobutyric acid (GABA; 10^-5–10^-4 M), and for the catecholamines adrenaline and noradrenaline, both in concentrations between 10^-5 and 10^-3 M. Serotonine (10^-5–10^-3 M) and dopamine (10^-5-10^-3 M) had no effect on resting or evoked activity in the Lorenzinian ampulla afferents. Acetylcholine (ACh; 10^-4 M) enlarged discharge frequency in those units with initial rates lower than 22–25 Hz, but diminished discharge frequency in fibres with initial activity higher than 25 Hz. When synaptic transmission was blocked by high Mg²⁺ solution, perfusion with additional ACh did not restore resting activity in the afferent fibre. The results suggest that the most probable transmitter in the afferent synapse of the ampullae of Lorenzini is l-GLU or l-ASP, or a substance of similar nature.Abbreviations ACh acetylcholine - GABA gamma aminobutyric acid - KA kainic acid - l-ASP l-aspartate - l-GLU l-glutamate - NMDA N-methyl-D-aspartate - Q quisqualate - n.s. normal solution     

The aerobic growth and product formation of Lactobacillus,Leuconostoc, Brochothrix,and Carnobacterium in batch cultures

Summary The aerobic growth and metabolism of eleven homofermentative and three heterofermentative Lactobacillus strains, three Leuconostoc strains, two Brochothrix thermosphacta strains and two Carnobacterium strains were studied in batch cultures at pH 6.0 and 25°C on a complex substrate containing 10.0 g glucose per litre. All strains, except Carnobacterium divergens 69, grew well aerobically. An oxygen consumption was registered for 18 of the strains—the exceptions being Lactobacillus alimentarius DSM 20249^T, Lactobacillus farciminis DSM 20284^T and Lactobacillus sharpeae DSM 20505^T. The homofermentative lactobacilli showed a maximal oxygen consumption during the stationary growth phase and this was coupled with a low final viable count. Leuconostoc strains, heterofermentative lactobacilli, Brochothrix thermosphacta and Carnobacterium strains showed a maximal oxygen consumption during the exponential growth phase together with a high final viable count. The maximum specific growth rate varied from 0.19 to 0.54 h^-1 while the growth yield varied from 19 to 86 g dry weight per mol glucose consumed. In general, homofermentative lactobacilli produced dl-lactic acid, acetic acid and acetoin. The three heterofermentative lactobacilli produced dl-lactic acid and acetic acid, two strains also produced ethanol Leuconostoc spp. formed d-lactic acid, acetic acid, and ethanol. B. thermosphacta produced acetoin, acetic acid, formic acid, isobutyric acid and isovaleric acid but no lactic acid. Carnobacterium produced l-lactic acid, acetic acid and acetoin. All strains accumulated hydrogen peroxide except L. alimentarius DSM 20249^T, Carnobacterium piscicola 3 and B. thermosphacta.née Blickstad     

Chemotaxonomic studies ofAsplenium sect.Hymenasplenium (Aspleniaceae)

We previously isolated and characterized a new free amino acid withd-configuration at the α-carbon,trans-3, 4-dehydro-d-2-aminopimelic acid and its related amino acids,d-2-aminopimelic acid and 4-hydroxy-l-2-aminopimelic acid fromAsplenium unilaterale. In this paper, we report that the biosynthetic relationshps among these three amino acids were studied using¹⁴C-and³H-labeled compounds as tracers. Glutamate and aspartate were shown to be good precursors and it was suggested that 4-hydroxy-l-2-aminopimelic acid is biosynthesized first and the twod-amino acids are derived from it. Furthermore, the distribution patterns of these non-protein amino acids inAsplenium sect.Hymenasplenium were examined in detail and they were evaluated by their biosynthetic pathway. Morphological characters especially on their rhizomes were also examined and their character phylogeny was determined by outgroup comparison. Taking all the characters available into account, the phylogenetic relationship among 7 species ofAsplenium sect.Hymenasplenium in Japan and Taiwan is discussed by the transformed cladistic method.     

The enzymes involved in the synthesis of phytic acid in Lemna gibba (Studies on the biosynthesis of cyclitols,XL.)

Summary The biosynthesis of phytic acid is known to be catalyzed by enzymes causing a stepwise phosphorylation of myo-inositol or 1l-myo-inositol 1-phosphate with adenosine triphosphate as phosphate donor. The kinases responsible for these phosphorylations in Lemna gibba were purified by affinity chromatography on a Sepharose gel carrying myo-inositol 2-phosphate at the binding site. Three fractions with enzymatic activity could be identified; in the first one, we find myo-inositol kinase (EC 2.7.1.64) phosphorylating myo-inositol to 1l-myo-inositol 1-phosphate; the second one brings about the phosphorylation of myo-inositol trisphosphate to phytic acid; the third one phosphorylates myo-inositol 1-phosphate to a myo-inositol trisphosphate. An enzyme oxidizing 1l-myo-inositol 1-phosphate to an uronic acid derivative is found in the first two fractions. In the presence of ATP, Mg²⁺ Mn²⁺, and the second and the third enzyme fractions in an appropriate mixture, 1l-myo-inositol 1-phosphate can be phosphorylated to phytic acid. The structure of the trisphosphate acting as an intermediate is not yet known.     

Functional characteristics ofl-glutamate,N-methyl-d-aspartate and kainate receptors in isolated brain synaptic membranes

l-Glutamic acid (l-Glu) and other excitatory amino acids and amino acid analogs enhanced [³⁵S]thiocyanate (SCN^–) uptake in isolated-resealed synaptic membrane vesicles. The SCN^– uptake was used as a measure of membrane depolarization to evaluate the characteristics of functional excitatory amino acid receptors in the synaptic membranes.N-Methyl-d-aspartate (NMDA) andl-Glu produced additive effects on SCN^– accumulation indicating the presence of distinctl-Glu and NMDA receptors. On the other hand, kainic acid (KA) andl-Glu shared either common receptor sites or ion channels. The effects of antagonists on NMDA,l-Glu, and KA stimulation of SCN^– influx were consistent with previously reported electrophysiologic observations in intact neurons.     

Nω-Hydroxyamino-α-amino acids as a new class of very strong inhibitors of arginases

 The effects of various compounds bearing an N-OH group such as N-hydroxy-guanidines, amidoximes, and hydroxylamines, on bovine and rat liver arginases was studied. Some of these compounds with an l-α-amino acid function at an appropriate distance from the N-OH group acted as strong competitive liver arginase inhibitors, displaying Ki values between 4 and 150 μM. Two compounds, N ^ε-hydroxy-l-lysine and N ^ω-hydroxy-d,l-indospicine, which exhibited Ki values of 4 and 20 μM (at pH 7.4), were the most potent inhibitors of arginase described to date. The distance between the α-amino acid and N-OH functions appeared to be crucial for potent inhibition of arginase, as N ^δ-hydroxy-l-ornithine, which has one -CH₂ group less than N ^ε-hydroxy-l-lysine, exhibited a 37-fold higher Ki value than N ^ε-hydroxy-l-lysine. Based on these results, a model for the interaction of N ^ω-hydroxyamino-l-α-amino acids with the arginase active site is proposed. This model involves the binding of the N-OH group of the inhibitors to the arginase Mn(II) center and suggests that N ^ε-hydroxy-l-lysine is a good transition state analog of arginase.     

α-Chymotrypsin-Catalyzed Peptide Synthesis Using <Emphasis Type="Italic">N</Emphasis>-Protected <Emphasis Type="SmallCaps">D</Emphasis>-Amino Acid Carbamoylmethyl Esters as Acyl Donors

α-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml⁻¹; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h.     

N5-(l-1-Carboxyethyl)-l-ornithine: NADP+ oxidoreductase inStreptococcus lactis: Distribution,constitutivity, and regulation

N⁵-(l-1-Carboxyethyl)-l-ornithine: NADP⁺ oxidoreductase [N⁵-(CE)ornithine synthase] catalyzes the NADPH-dependent reductive condensation between pyruvic acid and the terminal amino group ofl-ornithine andl-lysine to yield N⁵-(l-1-carboxyethyl)-l-ornithine and N⁶-(l-1-carboxyethyl)-l-lysine respectively. Polyclonal antibodies against N⁵-(CE)ornithine synthase purified fromStreptococcus lactis K1 have been used for the immunochemical (Western blot) detection and sizing of this enzyme in various lactic acid bacteria. The enzyme was confined to about one-half of the strains ofS. lactis examined. N⁵-(CE)ornithine synthase is constitutive, and in strains K1, 6F3, and (plasmid-free)H₁-4125 the native enzyme is a tetramer composed of identical subunits of M_r=38,000. However, in other strains, including 133 (ATCC 11454), C₁₀, and ML₈, the molecular weight of the native enzyme is approximately 130,000 and the corresponding subunit M_r=35,000. Analyses of the amino acid pool components maintained byS. lactis K1 during growth in medium containing [¹⁴C] labeled and unlabeled arginine have revealed that (i) exogenous arginine is the precursor of intracellular ornithine, citrulline, and N⁵-(CE)ornithine, and (ii) the rates of turnover of ornithine and citrulline were considerably faster than that of N⁵-(CE)ornithine. These data account for the biosynthesis and accumulation of N⁵-(CE)ornithine byS. lactis.     

Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-¹⁴C]d-(–)-hydroxybutyric acid, [1-¹⁴C]d-lactic acid, [1-¹⁴C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(–)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-¹⁴C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-¹⁴C]lactyl-CoA was used as a substrate at a rate that was only less than 10^–4 of the rate than with [3-¹⁴C]d-(–)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-¹⁴C]d-lactyl-CoA as substrate at a relatively high rate. Correspondence to: A. Steinbüchel     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by <Superscript>1</Superscript>H-nuclear magnetic resonance spectroscopy

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. ¹H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the ¹H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) ¹H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D ¹H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains. The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.     

l-Ascorbic-acid biosynthesis in the euryhaline diatom Cyclotella cryptica

l-Ascorbic acid (AA) production in cells of Cyclotella cryptica Reimann, Lewin, Guillard (Bacillariophyceae) is enhanced when darkadapted cells are exposed to light.Heterotrophically grown cells incubated with d-[6-³H,6-¹⁴C]glucose and d-[1-³H,6-¹⁴C]glucose (2 h in dark followed by 15 h light) produced labeled AA with significantly different ratios of ³H and ¹⁴C. Comparisons of labeling patterns in AA and chitin-derived d-glucosamine support a path of conversion in Cyclotella from d-glucose to AA that inverts the carbon chain of the sugar. This process resembles similar conversions found in AA-synthesizing animals and species from two other algal classes.Abbreviations AA l-Ascorbic acid - glc d-glucose - glcN d-glucosamine