A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

L-Gln and L-Ser suppress the D-galactosamine-induced IL-18 expression and hepatitis

d-Galactosamine (GalN) induces acute hepatitis in experimental animals and this hepatitis has been shown to be suppressed by preceding ingestion of amino acids such as Gly, l-Ser, and l-Gln. However, little is known about the mechanism of its action. The present study shows for the first time that IL-18 reduction is involved in the suppressive actions of l-Gln and l-Ser on GalN-induced hepatitis. Elevation of IL-18 mRNA expression in liver and its concentration in serum in GalN-treated rats were found to be suppressed by preceding ingestion of 10% l-Gln- or 10% l-Ser diets, and resulted in the attenuation of the increase in serum transaminase (ALT and AST) activities, indexes of hepatic injury. These results suggest that suppressive effects of some dietary amino acids on the GalN-induced hepatitis are mediated by IL-18 reduction.     

Oxidation of L-serine and L-threonine by bis(hydrogen periodato)argentate(III) complex anion: a mechanistic study

Oxidation of l-serine and l-threonine by a silver(III) complex anion, [Ag(HIO(6))(2)](5-), has been studied in aqueous alkaline medium. The oxidation products of the amino acids have been identified as ammonia, glyoxylic acid and aldehyde (formaldehyde for serine and acetaldehyde for threonine). Kinetics of the oxidation reactions has been followed by the conventional spectrophotometry in the temperature range of 20.0-35.0 degrees C and the reactions display an overall second-order behavior: first-order with respect to both Ag(III) and the amino acids. Analysis of influences of [OH(-)] and [periodate] on the second-order rate constants k' reveals an empirical rate expression: k(')=(k(a)+k(b)[OH(-)])K(1)/([H(2)IO(6)(3-)](e)+K(1)), where [H(2)IO(6)(3-)](e) is equilibrium concentration of periodate, and where k(a)=6.1+/-0.5M(-1)s(-1), k(b)=264+/-6M(-2)s(-1), and K(1)=(6.5+/-1.3)x10(-4)M for serine and k(a)=12.6+/-1.7M(-1)s(-1), k(b)=(5.5+/-0.2)x10(2)M(-2)s(-1), and K(1)=(6.2+/-1.5)x10(-4)M for threonine at 25.0 degrees C and ionic strength of 0.30M. Activation parameters associated with k(a) and k(b) have also been derived. A reaction mechanism is proposed to involve two pre-equilibria, leading to formation of an Ag(III)-periodato-amino acid ternary complex. The ternary complex undergoes a two-electron transfer from the coordinated amino acid to the metal center via two parallel pathways: one pathway is spontaneous and the other is assisted by a hydroxide ion. Potential applications of the Ag(III) complex as a reagent for modifications of peptides and proteins are implicated.     

Mechanisms of the protective effect of L-alanine to D-galactosamine-induced hepatocellular injury: comparative studies of L-alanine and pyruvate

The addition of L-alanine reduced lactate dehydrogenase leakage from primary cultured rat hepatocytes treated with galactosamine (D-gal), while D-alanine and other amino acids did not. However, the mechanisms have not yet been entirely clarified. In this study, we used various inhibitors of metabolism, i.e., aminooxyacetate, oligomycin, and quinolinic acid, to examine the relation between this protective effect and the metabolism of L-alanine. Quinolinic acid (10 mM) did not affect the hepatoprotective effect of L-alanine, while oligomycin (0.1 mug/ml) and aminooxyacetate (1 mM) eliminated the hepatoprotective effect of L-alanine. L-Alanine also increased the albumin secretion by cultured hepatocytes treated with D-gal, while pyruvate had little effect. It was revealed that the intracellular content of pyruvate did not increase as a result of addition of L-alanine. These results are consistent with the hypothesis that L-alanine metabolism is important for hepatoprotection, but pyruvate cannot be used as a substitute for L-alanine.     

Effect of Dietary l-Glutamine on the Hepatotoxic Action of d-Galactosamine in Rats

The protective effect of dietary l-glutamine against the hepatotoxic action of d-galactosamine (GalN) was investigated by model experiments with rats. Rats fed with 20% casein diets containing 10% free amino acids were injected with GalN, and the serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activities and the hepatic glycogen content were assayed 20 hours after the injection. These enzyme activities in the group fed with the 10% l-glutamine diet for 8 days were lower than those in the groups fed with the control, 10% l-glutamic acid and 10% l-alanine diets for 8 days. The more prolonged the feeding period with the 10% l-glutamine diet was, the more the serum activity levels of such enzymes were decreased. Although neomycin also lowered these enzyme activities, its simultaneous ingestion with neomycin did not show any additive or synergistic effect. The hepatic glycogen content in the 10% glutamine group still remained high after the GalN treatment. It is therefore assumed that the effectiveness of glutamine intake would have been mediated by glycogen metabolism rather than by uridine metabolism.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Epitope mapping of human aromatic L-amino acid decarboxylase

Autoimmune polyendocrine syndrome type I (APS I) is a rare hereditary condition considered a model disease for organ specific autoimmunity. A wide range of autoantibodies targeting antigens present in the affected organs have been identified. Autoantibodies against aromatic L-amino acid decarboxylase (AADC) are present in about 50% of APS I patients. In order to increase our understanding of autoantibody specificity in APS I, the aim of the present study was to localize target regions on AADC recognized by sera from APS I patients. Using several complementing strategies, we have shown that autoantibodies against AADC mainly recognize conformational epitopes. The major antigenic determinants were detected N-terminally to amino acid residue 237. Replacement of amino acids 227-230 (ERDK) with alanine residues reduced the reactivity towards AADC by >80% in all patient sera tested, suggesting that amino acids 227-230 are an important part of an immunodominant epitope.     

D-Erythroascorbic acid activates cyanide-resistant respiration in Candida albicans

Higher plants, protists and fungi possess cyanide-resistant respiratory pathway, which is mediated by alternative oxidase (AOX). The activity of AOX has been found to be dependent on several regulatory mechanisms including gene expression and posttranslational regulation. In the present study, we report that the presence of cyanide in culture medium remarkably retarded the growth of alo1/alo1 mutant of Candida albicans, which lacks d-arabinono-1,4-lactone oxidase (ALO) that catalyzes the final step of d-erythroascorbic acid (EASC) biosynthesis. Measurement of respiratory activity and Western blot analysis revealed that increase in the intracellular EASC level induces the expression of AOX in C. albicans. AOX could still be induced by antimycin A, a respiratory inhibitor, in the absence of EASC, suggesting that several factors may act in parallel pathways to induce the expression of AOX. Taken together, our results suggest that EASC plays important roles in activation of cyanide-resistant respiration in C. albicans.     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of l-threonine

l-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize l-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of l-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including l-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined l-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.     

Achacin induces cell death in HeLa cells through two different mechanisms

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.     

High-throughput determination of free D-aspartic acid in mammals by enzyme immunoassay using specific monoclonal antibody

A method for rapid determination of free d-aspartic acid (d-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against d-Asp for the first time. An anti-d-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated d-Asp to BALB/c mice. The obtained antibody has a high specificity toward d-Asp but shows a slight cross-reactivity to all other d- and l- amino acids including l-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 μmol/mL d-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of d-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of d-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of d-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this d-amino acid.     

Volatile Compounds Produced by the Reaction of L-Cysteine or L-Cystine with Carbonyl Compounds

Sulfur-containing amino acids (L-cysteine or L-cystine) were reacted with D-glucose or pyruvaldehyde at various temperatures and submitted to flavor evaluation. The nuance of the aroma was changed with temperature, and the most acceptable aroma (Japanese rice cracker with sesame-like) was produced at 160°C in all the samples. Volatile compounds produced at 160°C were investigated by gas chromatography and GC–MS coupling. Many compounds such as thiazoles and thiophenes found in the volatiles of some foodstuffs were identified.     

A highly specific glyoxylate reductase derived from a formate dehydrogenase

A Glu141Asn mutant Paracoccus sp. 12-A formate dehydrogenase catalyzes marked glyoxylate reduction. Additional replacement of the His332-Gln313 pair with His-Glu, which is a consensus acid/base catalyst in D-hydroxyacid dehydrogenases, further improved the catalytic activity of the enzyme as to glyoxylate reduction through enhancement of the hydrogen transfer step in the catalytic process, slightly shifting the optimal pH for the reaction. On the other hand, the replacement induced no marked activity toward other 2-ketoacid substrates, and diminished the enzyme activity as to formate oxidation. Consequently, the formate dehydrogenase was converted to a highly specific and active glyoxylate reductase through only the two amino acid replacements.     

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809.     

Binding of different monosaccharides by lectin PA-IIL from Pseudomonas aeruginosa: thermodynamics data correlated with X-ray structures

The lectin from Pseudomonas aeruginosa (PA-IIL) is involved in host recognition and biofilm formation. Lectin not only displays an unusually high affinity for fucose but also binds to L-fucose, L-galactose and D-arabinose that differ only by the group at position 5 of the sugar ring. Isothermal calorimetry experiments provided precise determination of affinity for the three methyl-glycosides and revealed a large enthalpy contribution. The crystal structures of the complexes of PA-IIL with L-galactose and Met-beta-D-arabinoside have been determined and compared with the PA-IIL/fucose complex described previously. A combination of the structures and thermodynamics provided clues for the role of the hydrophobic group in affinity.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

The presence of high concentrations of free D-amino acids in human saliva

Free neutral D-amino acids have previously been detected in human plasma, usually accounting for less than 2% of the total free amino acid concentration (D-amino acid ratio) [Nagata, Y., Masui, R., Akino, T., 1992a. The presence of free D-serine, D-alanine and D-proline in human plasma. Experientia 48, 986-988. Nagata, Y., Yamamoto, K., Shimojo, T., 1992b. Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography. Journal of Chromatography 575, 147-152. Nagata, Y., Yamamoto, K., Shimojo, T., Konno, R., Yasumura, Y., Akino, T., 1992c. The presence of free D-alanine, D-proline and D-serine in mice. Biochimca et Biiophysica Acta 1115, 208-211]. In the present study to search for the source of free D-amino acids, D- and L-enantiomers of the major non-essential amino acids, i.e., the free form of serine, alanine, proline, aspartate and glutamate were analyzed by HPLC in human saliva, submandibular glands and oral epithelial cells. The D-enantiomer ratios to total of free alanine or proline were 35% and 20%, respectively, in saliva. The ratios of the other D-amino acids were less than 11%. The effect of ingested food and oral bacteria on the saliva amino acid levels was suggested to be insignificant. D-Alanine and d-aspartate were also detected in the submandibular gland in ratios up to 5%, and D-alanine and d-proline were found in oral epithelial cells in ratios of 18% and 5%, respectively. The submandibular gland and oral epithelial cells are suggested to be possible sources of the saliva D-alanine and D-aspartate.     

Purification and Characterization,of Rice Bran Lipase II

A species of rice bran lipase (lipase II) was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose, Sephadex G–75 and CH-Sephadex C–50. Both polyacrylamide disc electrophoresis and ultracentrifugation demonstrated that the enzyme protein is homogeneous. The isoelectric point of the enzyme was 9.10 by ampholine electrophoresis. The sedimentation coefficient of the enzyme was evaluated to be 2.60 S, and the molecular weight to be 33,300 according to Archbald’s method. The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 27°C. It was stable over the pH range from 5 to 9.5 and below 30°C. In substrate specificity, the enzyme exhibited a high specificity toward triglycerides having short-carbon chain fatty acids, although it was capable of hydrolyzing the ester bonds in the rice and olive oil.