Origin ofd-serine present in urine of mutant mice lackingd-amino-acid oxidase activity

Summary Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO⁺ mice. Most of the serine wasd-isomer. The origin of this^d-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO^- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO^- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO^- mice still contained 4.6 times mored-serine than that of the ddY/DAO⁺ mice, suggesting a part of the D-serine have an endogenous origin.     

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.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.     

d-Aminoaciduria in mutant mice lackingd-amino-acid oxidase activity

Summary Urine of mutant ddY/DAO^– mice lackingd-amino-acid oxidase activity contained more serine and proline than that of normal ddY/DAO⁺ mice.d-Amino-acid oxidase treatment of urinary amino acids decreased the serine and proline, suggesting that they containedd-isomers. An HPLC analysis confirmed the presence ofd-serine. Urinary serine and proline contents were not decreased when the ddY/DAO^– mice were fed a diet which did not contain supplementaryd-methionine or when they were given water containing antibiotics. These results suggest that thed-serine andd-proline do not derive from thed-methionine supplemented in the diet or from intestinal bacteria. In urine of the ddY/DAO^– mice, a substance which seemed to bed-methionine sulfoxide and/ord-methionine sulfone was present. It is probably a metabolite of thed-methionine supplemented in the diet. Thed-aminoaciduria in the mutant mice lackingd-amino-acid oxidase activity indicates that this enzyme is involved in the metabolism of thed-amino acids in normal mice.     

d-Glucose does not catabolite repress a transketolase-deficientd-ribose-producingBacillus subtilis mutant strain

WhenBacillus subtilis strain ATCC 21951, a transketolase-deficientd-ribose-producing mutant, was grown ond-glucose plus a second substrate which is metabolized via the oxidative pentose phosphate cycle (d-gluconic acid,d-xylose,l-arabinose ord-xylitol),d-glucose did not catabolite repress metabolism of the second carbon source. Thed-ribose yield obtained with the simultaneously converted carbon substrates, significantly exceeded that when onlyd-glucose was used. In addition, the concentration of glycolytic by-products and the fermentation time significantly decreased. Based on these findings, a fermentation process was developed withB. subtilis strain ATCC 21951 in whichd-glucose (100 g L^–1) andd-gluconic acid (50 g L^–1) were converted into 45 g L^–1 ofd-ribose and 7.5 g L^–1 of acetoin. A second process, based ond-glucose andd-xylose (100 g L^–1 each), yielded 60 g L^–1 ofd-ribose and 4 g L^–1 of acetoin plus 2,3-butanediol. Both mixed carbon source fermentations provide excellent alternatives to the less efficientd-glucose-based processes used so far.     

The alternative<Emphasis Type="SmallCaps"> d</Emphasis>-galactose degrading pathway of<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis> proceeds via<Emphasis Type="SmallCaps"> l</Emphasis>-sorbose

The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD⁺-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD⁺-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.     

Incorporation of two18O atoms into a peptide during isoaspartyl repair reveals repeated passage through a succinimide intermediate

To study the mechanism of protein carboxyl methyltransferase-driven repair of age-damaged sites in polypeptides, a modell-isoaspartyl peptide,l-isotetragastrin, was enzymatically repaired to normall-tetragastrin in the presence of¹⁸O-enriched water. By this design, the enrichment of¹⁸O atoms in the peptide would reflect the number of passages through a hydrolyzable succinimide intermediate during formation of the repaired product. Mass determinations by FAB mass spectrometry revealed repaired peptide with two¹⁸O atoms incorporated, demonstrating that more than a single cycle of methylation and demethylation is necessary to ensure stoichiometric repair.Abbreviations HPLC high-pressure liquid chromatography - FAB fast atom bombardment - TFA trifluoroacetic acid - PCM proteind-aspartyl/L-isoaspartyl carboxyl methyltransfer-ase - l-Normal [l-Asp³]tetragastrin - l-Iso [L-isoAsp³]tetragastrin - d-Normal [d-Asp³]tetragastrin - d-Iso [d-isoAsp³]tetragastrin     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

d-Aspartyl residue in a peptide can be liberated and metabolized by pig kidney enzymes

Summary The presence of an enzyme activity which hydrolyzes glycyl-d-aspartate was found in the homogenates of pig kidney cortex. The activity was inhibited by metal chelating agents and cilastatin, suggesting that the enzyme was a cilastatin-sensitive metallo-peptidase. Of the two hydrolysis products,d-aspartate was found to be less accumulated than glycine. The fate ofd-aspartate was, therefore, examined and the amino acid was found to be converted tol-aspartate,l-alanine and pyruvate, in the presence ofl-glutamate. Experiments with enzyme inhibitors suggested that the conversion involvedd-aspartate oxidase, aspartate aminotransferase and alanine aminotransferase as well as decarboxylation of oxaloacetate produced fromd-aspartate. All the results indicate that the enzymes in the pig kidney can liberate thed-aspartyl residue in the peptide and convert it to the compounds readily utilizable. The finding suggests a probable metabolic pathway of thed-aspartate-containing peptide.     

Na+-independentl-aspartate binding sites in chick brain

1. One binding component with aK _d value of 200×10^–9 M and half-life of the ligand binding component of 30 min was found. 2. Chloride ions produced a significant increase ofl-[³H]aspartate andl-[³H]glutamate binding. 3.l-Glutamate,l-ibotenate,l-quisqualate, anddl-homocysteic acid were potent inhibitors ofl-[³H]aspartate binding. 4. In all brain regions major increases of binding were observed during the third week of the in ovo period of life.     

Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

Studies on isolated subcellular components of cat pancreas

Summary Transport of alanine was studied in isolated plasma membrane vesicles from cat pancreas using a rapid filtration technique. The uptake is osmotically sensitive and the kinetics ofl-alanine transport are biphasic showing a saturable and a nonsaturable component. The saturable component is seen only when a sodium gradient directed from the medium to the vesicular space is present. Under this condition an overshooting uptake ofl-but not ofd-alanine occurs. The Na⁺ gradient stimulated uptake ofl-alanine is inhibited byl-serine andl-leucine and stimulated when the membrane vesicles had been preloaded withl-alanine,l-serine orl-leucine.The ionophore monensin inhibits stimulation of uptake caused by a sodium gradient. In the presence of valinomycin or carbonyl cyanidep-trifluoromethoxyphenylhydrazone (CFCCP), the sodium-dependent transport is augmented in vesicles preloaded with K₂SO₄ or H⁺ ions (intravesicular pH 5.5), respectively. In the presence of different anions, the Na⁺-dependent transport is stimulated according to increasing anionic penetration through membranes (lipid solubility). We conclude that a sodium dependent electrogenic amino acid transport system is present in pancreatic plasma membranes.     

Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

l-tyrosine-binding proteins on melanoma cells

Summary Crosslinking of [¹⁴C]l-tyrosine to at least five hamster melanoma cell surface proteins is reported. This effect was abolished by addition of nonradioactivel-tyrosine,l-phenylalanine, orl-dopa, but not byd-tyrosine, tyramine, dopamine, norepinephrine, or epinephrine. The above proteins can be purified by tyrosine-affinity chromatography. They have molecular weights different from proteins staining for dopa oxidase and proteins that bind anti-tyrosinase antibody in Western blots. It is suggested that they may be a hithergo unrecognized part of the cellular apparatus governing melanogenesis.     

Xylose metabolism in the anaerobic fungus<Emphasis Type="Italic"> Piromyces</Emphasis> sp. strain E2 follows the bacterial pathway

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min^–1mg protein^-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

Up-regulation of d-serine Might Induce GABAergic Neuronal Degeneration in the Cerebral Cortex and Hippocampus in the Mouse Pilocarpine Model of Epilepsy

Epilepsy is a serious neurological disorder with neuronal loss and spontaneous recurrent seizures, but the neurochemical basis remains largely unclear. We hypothesize that d-serine, a newly identified endogenous co-agonist of N-methyl-d-aspartate (NMDA) receptor, may trigger excitotoxicity and neuronal damage in epileptogenesis. By using a mouse pilocarpine model, immunohistochemistry, Fluoro-Jade staining and double-labeling, the present study revealed up-regulation of d-serine expression in a proportion (41%) of neurons in the cerebral cortex and hippocampus. The d-serine-positive neurons occurred at 4 h, reached peak levels at 12–24 h, and gradually went down at 3–14 days. Moreover, most of d-serine-positive neurons were GABAergic (98%), underwent degenerating death (93%), and were accompanied enhancing phosphorylation of NMDA receptor subunit 1. This study has provided new evidence that up-regulation of d-serine production might induce GABAergic neuronal degeneration through excitotoxic mechanism in the pilocarpine model and may be involved in early pathogenesis and recurrent seizure of chronic epilepsy. Ms. L. Wang is on leave from Department of Neurology, Kunming General Hospital of Chengdu Military Region, China.