An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

Acetyl-l-carnitine influences the fluidity of brain microsomes and of liposomes made of rat brain microsomal lipid extracts

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) was measured in different preparations (bovine spinal cord phosphatidylserine liposomes, rat brain microsomes, liposomes made with rat brain microsomal lipid having different phospholipid:cholesterol ratios) at temperatures ranging from 10° to 55°C. Phosphatidylserine liposomes exhibited an exponential relationship of rversus temperature, whereas the relationship shown by microsomes and liposomes prepared with microsomal lipid extracts was a linear one. The removal of protein and high phospholipid:cholesterol ratios decreased the slope of the lines (fluidity increased), although the intercept was unaffected. This means that differences were better appreciated at high temperatures and were well evident at 37°C. Acetyl-l-carnitine decreased r in rat brain microsomes and in liposomes made with microsomal lipids with different phospholipid:cholesterol ratios. The fluidifying effect of acetyl-l-carnitine was mild but statistically significant and could explain, at least in part, the data reported in the literature of acetyl-l-carnitine acting on some parameters affected by ageing. Besides, acetyl-l-carnitine seemed to oppose the changes of viscosity due to lipid peroxidation, which has been reported to increase in ageing and dementia.l-carnitine shares the properties of its acetyl ester, but only in part.Abbreviations DPH diphenylhexatriene - HEPES 4-(2-hydroxyethyl-l-piperazineethansulfonic) acid - r fluorescence anisotropy - SHB sucrose-HEPES-buffer (0.32 M sucrose, 2 mM HEPES, pH 7.0)     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

Astrocytes as potential modulators of mercuric chloride neurotoxicity

Summary 1. MC has been shown to inhibit the uptake ofl-glutamate and increased-aspartate release from preloaded astrocytes in a dose-dependent fashion.2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-inducedd-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5µM); when added after MC treatment, DTT fully inhibits the MC-inducedd-aspartate release, while GSH does not.3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocyticd-aspartate release. Other studies demonstrate that although MC treatment (5µM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate.4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocyticd-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca²⁺-channel antagonist nimodipine (10µM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.     

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.     

Visible wavelength spectrophotometric assays of l-aspartate and d-aspartate using hyperthermophilic enzyme systems

Methods with which to simply and rapidly assay l-aspartate (l-Asp) and d-aspartate (d-Asp) would be highly useful for physiological research and for nutritional and clinical analyses. Levels of l- and d-Asp in food and cell extracts are currently determined using high-performance liquid chromatography. However, this method is time-consuming and expensive. Here we describe a simple and specific method for using an l-aspartate dehydrogenase (l-AspDH) system to colorimetrically assay l-Asp and a system of three hyperthermophilic enzymes—aspartate racemase (AspR), l-AspDH, and l-aspartate oxidase (l-AO)—to assay d-Asp. In the former, the reaction rate of nicotinamide adenine dinucleotide (NAD⁺)-dependent l-AspDH was measured based on increases in the absorbance at 438 nm, reflecting formation of formazan from water-soluble tetrazolium-1 (WST-1), using 1-methoxy-5-methylphenazinum methyl sulfate (mPMS) as a redox mediator. In the latter, d-Asp was measured after first removing l-Asp in the sample solution with l-AO. The remaining d-Asp was then changed to l-Asp using racemase, and the newly formed l-Asp was assayed calorimetrically using NAD⁺-dependent aspartate dehydrogenase as described above. This method enables simple and rapid spectrophotometric determination of 1 to 100 μM l- and d-Asp in the assay systems. In addition, methods were applicable to the l- and d-Asp determinations in some living cells and foods.     

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.     

Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-l-arginine methyl ester

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [³H]l-arginine into [³H]l-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.     

Determination of N-nitroso-l-arginine in rat brain extracts by capillary electrophoresis using photodiode-array detection

N-Nitroso-l-arginine was described as one of the products of l-arginine metabolism in biological media. A simple and rapid method to determine its concentration in rat brain was developed. Capillary electrophoresis with a photodiode-array detector was used at 254 nm, permitting the quantification of N-nitroso-l-arginine. The detection limit in biological solution was 1 μg/ml.     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.     

Effect of acetyl-l-carnitine on extracellular amino acid levels in vivo in rat brain regions

Acetyl-l-carnitine (ALCAR) was found to have beneficial effects in senile patients. In recent years many of its effects on the nervous system have been examined, but its mechanism(s) of action remains to be elucidated. We previously reported that it causes release of dopamine in the striatum. In the present paper we report that ALCAR, when administered at intracerebral sites via microdialysis, stimulates the release of amino acids in a concentration-dependent and regionally heterogeneous manner. The effect was strong in the striatum and cerebellum, less so in the frontal cortex, and weak in the thalamus. Seven amino acids were measured: the increase in the level of aspartate, glutamate, and taurine was substantial, and the increase in the level of glycine, serine, threonine, alanine, and glutamine in the microdialysate was minor. The stimulatory effect of ALCAR on the release of amino acids in the striatum was inhibited by the muscarinic antagonist atropine, but was not inhibited by the nicotinic antagonist mecamylamine. The effect of ALCAR on the levels of most of the amino acids tested was independent of the presence of Ca²⁺ in the perfused. These results indicate that ALCAR, when administered intracerebrally at fairly high concentrations, can affect the level and the release not only of such neurotransmitters as acetylcholine and dopamine, but also of amino acids. The mechanism of action of ALCAR on the release of cerebral amino acids may involve the participation of muscarinic receptors or may be mediated through the release of dopamine, but the lack of Ca²⁺ dependence indicates a release from the cytoplasmic amino acid pool, possibly through the effect of ALCAR on cell membrane permeability.     

Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus

In hippocampal slices arachidonic acid released after NMDA post-synaptic receptor activation is thought to act as a retrograde trans-synaptic messenger which facilitates the pre-synaptic release of L-glutamate to be involved in the expression of long-term synaptic potentiation (LTP). We measured the mass amount of arachidonic acid released from hippocampal slices incubated under conditions which maintain the electrophysiological responsiveness of the slice. Melittin released arachidonic, oleic and docosahexaenoic acids by phospholipase A₂ activation but not palmitic or stearic acids. Of greater interestl-glutamate, N-methyl-d-aspartate and incubation conditions known to induce LTP selectively and rapidly increased the release of archidonic acid in amounts over basal levels of 200–300 ng/mg protein. This is the first direct determination of the mass amount of arachidonic acid released following NMDA receptor activation in the hippocampus.Special issue dedicated to Dr. Louis Sokoloff.     

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     

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.     

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.     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Synthesis and application of dipeptides; current status and perspectives

The functions and applications of l-α-dipeptides (dipeptides) have been poorly studied compared with proteins or amino acids. Only a few dipeptides, such as aspartame (l-aspartyl-l-phenylalanine methyl ester) and l-alanyl-l-glutamine (Ala-Gln), are commercially used. This can be attributed to the lack of an efficient process for dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however, novel methods have arisen for dipeptide synthesis including a nonribosomal peptide-synthetase-based method and an l-amino acid α-ligase-based method, both of which enable dipeptides to be produced through fermentative processes. Since it has been revealed that some dipeptides have unique physiological functions, the progress in production methods will undoubtedly accelerate the applications of dipeptides in many fields. In this review, the functions and applications of dipeptides, mainly in commercial use, and methods for dipeptide production including already proven processes as well as newly developed ones are summarized. As aspartame and Ala-Gln are produced using different industrial processes, the manufacturing processes of these two dipeptides are compared to clarify the characteristics of each procedure.     

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.