The effects of two anticonvulsants on amino acid levels in the developing rat cerebellum

Two anticonvulsants were administered pre- and postnatally to determine their effects on putative amino acid neurotransmitter levels in the rat cerebellum. The amino acids were quantitated using precolumn fluorescence derivatization and reverse-phase high performance liquid chromatography at various postnatal intervals. Treatment with clonazepam produced an initial depression in levels of most of the amino acids analyised. By three weeks postnatal all the amino acids, with the exception of GABA, had returned to control levels. GABA levels were still depressed five weeks after the cessation of treatment. Phenobarbital treatment produced an initial elevation in the level of GABA. At three weeks postnatal, both GABA and glutamate levels were elevated and remained so at eight weeks postnatal. In conclusion, the data demonstrated that each anticonvulsant produced unique, acute and chronic alterations in the levels of the cerebellar amino acids.     

Characterization and expression of L-amino acid oxidase of mouse milk

l-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with l-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His other 11 amino acids tested and produced H(2)O(2) in a dose- and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N- and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H(2)O(2) using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.     

食用真菌发酵豆浆过程中对豆浆中几种生物活性物质的影响

本实验以灵芝、金耳以及冬虫夏草作为发酵菌种,选用豆浆为基本原料配制培养基,在发酵过程中定时取样并测定其中几种生物活性物质的含量,分析各生物活性物质在发酵过程中的变化。研究结果表明,三种食用真菌发酵豆浆的发酵液中总游离型大豆异黄酮含量在发酵过程中均缓慢上升并分别在发酵72 h、24 h和36 h达到最高,与发酵初期相比分别增加了31.81μg/m L、19.05μg/m L和15.71μg/m L;三种食用真菌发酵液中蛋白质含量随着发酵时间的推移而降低的同时氨基酸含量均有增加,且发酵液中的肽主要是分子量范围在300 Da~1 500 Da之间的具有较高生物活性的小分子肽,发酵液中的必需氨基酸含量以及占总氨基酸含量的比值均有增加。本实验探究了食用真菌发酵豆浆过程中生物活性物质的变化,为日后的富集游离型大豆异黄酮、多肽以及氨基酸等生物活性物质实验提供依据,并为工业化生产提供实践基础。     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Isolation of γ-aminobutyric acid-producing bacteria and optimization of fermentative medium

Ten γ-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) strains were isolated from kimchi and yoghurt. The strain B, isolated from kimchi showed the highest GABA-producing ability (3.68 g/L) in MRS broth with 1% monosodium glutamate (MSG). Strain B was identified as Lactococcus lactis subsp. lactis. The GABA-producing ability of L. lactis B was investigated using brown rice juice, germinated soybean juice and enzymolyzed skim milk as medium compositions. The D-optimal mixture design was applied to optimize the ratio of the three kinds of components in the media. The results showed that when the mixing ratio of brown rice juice, germinated soybean juice and enzymolyzed skim milk was 33:58:9 (v:v:v), the maximum GABA yield of L. lactis B was 6.41 g/L.     

Effect of transient neonatal hypothyroidism on the free aspartic acid, glutamic acid, and GABA content of different central auditory regions in the rat

The aspartic acid, glutamic acid, and gamma-aminobutyric acid (GABA) contents were determined in four central auditory system regions in rats with transient neonatal hypothyroidism compared with control ones: the ventral and dorsal parts of the cochlear nucleus, the central nucleus of the inferior colliculus, the auditory cortex, and in an extra-auditory structure, the substantia nigra pars reticulata. The animals were sacrificed at 50 days of age, brain tissue samples were taken out by microdissection, and the free amino acids were extracted. The amino acid content was assessed by double-isotope labelling following two-dimensional thin-layer chromatography separation. GABA content was significantly decreased in both cochlear nucleus regions and glutamic acid was elevated in the inferior colliculus. Neonatal hypothyroidism had no significant effect on the aspartic acid levels in the regions studied. The results suggest an effect of neonatal hypothyroidism on regional contents of free amino acids known as candidate neurotransmitters in the auditory system.     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Stimulation of gamma-aminobutyric acid synthesis activity in brown rice by a chitosan/glutamic acid germination solution and calcium/calmodulin

Changes in the concentrations of gamma-aminobutyric acid (GABA), soluble calcium ions, glutamic acid, and the activity of glutamate decarboxylase (GAD) were investigated in non-germinated vs. germinated brown rice. Brown rice was germinated for 72 h by applying each of the following solutions: (1) distilled water, (2) 5 mM lactic acid, (3) 50 ppm chitosan in 5 mM lactic acid, (4) 5 mM glutamic acid, and (5) 50 ppm chitosan in 5 mM glutamic acid. GABA concentrations were enhanced in all of the germinated brown rice when compared to the non-germinated brown rice. The GABA concentration was highest in the chitosan/glutamic acid that germinated brown rice at 2,011 nmol/g fresh weight, which was 13 times higher than the GABA concentration in the non-germinated brown rice at 154 nmol/g fresh weight. The concentrations of glutamic acid were significantly decreased in all of the germinated rice, regardless of the germination solution. Soluble calcium and GAD were higher in the germinated brown rice with the chitosan/glutamic acid solution when compared to the rice that was germinated in the other solutions. GAD that was partially purified from germinated brown rice was stimulated about 3.6-fold by the addition of calmodulin in the presence of calcium. These data show that the germination of brown rice in a chitosan/glutamic acid solution can significantly increase GABA synthesis activity and the concentration of GABA.     

The establishment of essential and metabolically derived amino acid profiles in developing chick embryo organs

The accumulation of certain essential and metabolically derived amino acids in the free amino acid pools of three excitable tissues has been studied in the chick embryo. Valine together with leucine are at the onset present in the yolk at higher concentrations than any of the other essential amino acids. By 15 days all the amino acids studied have accumulated in the embryonic pools at a higher rate than valine, although certain amino acids, such as phenylalanine or methionine, always remain at lower relative concentrations than valine. This reflects their low supply in the yolk, rather than a more rapid rate of disappearance (utilization). During early embryogenesis (E₂–E₄), tissues preferentially concentrate glutamic acid, besides taurine and phosphoethanolamine (6). The next distinct stage of development (E₄–E₇) is marked in the brain by a gradual rise in glutamic acid, glutamine and aspartic acid; the same three amino, acids do not demonstrate a further increase in the pool of the heart, while in the whole eye the amino acid profile begins to resemble the blood. Leucine in all three tissues declines rapidly, to reach isoleucine levels by day 7 of development; tyrosine increases slowly in apparent reciprocity to an equally gradual phenylalanine decrease. Into the second week of embryo growth (E₇–E₁₅), GABA appears in the mesencephalon (E₇) and the eye (E₉–E₁₀). In the mesencephalon, the free amino acid pool composition exhibits a rather sudden increase of most metabolically derived amino acids. Glutamic acid and glutamine in the brain increase in parallel; the rate of GABA and aspartic acid accumulation is slower, and for GABA stabilizes on day 14, as does glutamine. In the eye, by day 15, GABA levels are more closely aligned with the aspartic acid content. Finally, throughout embryogenesis serine fluctuations in blood and tissues are in parallel with those of threonine, and different from glycine or alanine which also change in tandem.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).     

Influence of the soya meal fractions on gibberellic acid and gibberellin A production in submerse cultivation ofGibberella fujikuroi

The relationship of different soya meal components to gibberellin (GA) production was studied. Fluorometric assay confirmed that under the given fermentation conditions, only gibberellic acid (GA₃) was synthesized on medium containing corn steep. On substituting soya flour for corn steep, the same amount of GA₃ was produced and in addition gibberellin A (GA₁) was formed. The GA₃: GA₁ ratio was 1∶1. The course of fermentation in media containing the soya meal protein fraction (fraction I), the soya meal amino acid complex, the corn steep amino acid complex and individual amino acids (γ-aminobutyric acid or tryptophane) was the same as in the control medium containing soya meal. The soya meal fraction II, which is characterized by a high cellulose and carbohydrate content, raised GA production by 25% as compared with production in medium containling soya meal; it simultaneously stimulated GA₃ production, so that the final GA₃: GA₁ ratio was 4∶1.     

Liquid chromatographic determination of gamma-aminobutyric acid in cerebrospinal fluid using 2-hydroxynaphthaldehyde as derivatizing reagent

gamma Aminobutyric acid (GABA) was determined by precolumn derivatization with 2-hydroxynaphthaldehyde and elution was made using Phenomenex C(18), 5 microm column with methanol: water (62:38 v/v) and UV detection at 330 nm. In a mixture containing glycine, l-lysine and tyramine GABA separated completely. A number of amines and amino acids tested did not affect the response of GABA. A linear calibration curve was obtained for GABA in the range of 1.2-28.0 microg/ml with detection limit of 2.8 ng/injection (5 microl). The method was used for the determination of GABA in cerebral spinal fluid (CSF) samples and gave results of 19.0 to 22.4 microg/m1 with coefficient of variation 2.4%     

Amino acid profiles in long-evans rat superior colliculus,visual cortex,and inferior colliculus

An ultransensitive triple-column ion-exchange/fluorometric method was utilized to measure the levels of over 30 amino acids and related primary amino compounds in Long-Evans rat superior colliculus (SC), visual cortex (VC) and inferior colliculus (IC). Comparison of levels of amino compounds revealed distinctly different profiles for each region. Major constituents were the neurotransmitters and related compounds glutamate, glutamine, GABA, taurine, aspartate and glycine. Glutathione levels were also relatively high in all three regions. SC exhibited a significantly higher level of GABA and -alanine compared to both VC and IC. VC had significantly higher levels of glutamate and taurine. VC exhibited the lowest level of glycine and IC the highest. A time-course experiment using SC documented that levels of eleven of thirty-four compounds, including GABA, were subject to significant postmortem alteration in vitro. SC GABA stability experiments indicated that significant in vitro increases of free GABA levels between 1 and 4 min postmortem were associated with equimolar decreases of conjugated GABA levels.     

Free Radicals and the Ischemia-Evoked Extracellular Accumulation of Amino Acids in Rat Cerebral Cortex

The effects of free radical generating systems on basal and ischemia/reperfusion-evoked release of amino acids into cortical superfusates was examined in the rat using the cortical cup technique. Xanthine oxidase plus xanthine significantly enhanced GABA levels 358 fold over controls during 20 min of four vessel occlusion. Glutamate and phosphoethanolamine release following reperfusion were also elevated. Prostaglandin synthase plus arachidonic acid significantly enhanced the ischemia-evoked release of all amino acids (aspartate 360 fold; glutamate 433 fold; glycine 6 fold; GABA 689 fold; phosphoethanolamine 69 fold) and increased the pre-ischemic levels of glutamate, glycine and phosphoethanolamine. Administration of H₂O₂ plus ferrous sulfate significantly elevated both pre-ischemic amino acid release and ischemia-evoked release. A role for free radical generating systems in the development of ischemic injury is supported by the ability of superoxide dismutase plus catalase to reduce ischemia-evoked amino acid efflux into cortical superfusates. Thus, the species of free radical produced, as well as the amount generated, may alter the pattern of amino acid release under both ischemic and non-ischemic conditions.     

In silico analysis of amino acid biosynthesis and proteolysis in Lactobacillus delbrueckii subsp. bulgaricus 2038 and the implications for bovine milk fermentation

The amino acid biosynthesis pathway and proteolytic system of Lactobacillus delbrueckii subsp. bulgaricus 2038 (L. bulgaricus 2038), a mainstay of large-scale yogurt production, were modeled based on its genomic sequence. L. bulgaricus 2038 retains more potential for amino acid synthesis and a more powerful proteolytic system than other L. bulgaricus strains, but favors amino acid uptake over de novo synthesis. Free amino acids and peptides in bovine milk provide the main nitrogen sources; whey is more important than casein for L. bulgaricus during fermentation. Free amino acids are imported by amino acid permeases and by ABC-type transport systems whereas exogenous oligopeptides are imported by ABC-type proteins only. Histidine is neither synthesized nor imported singly, which might explain why L. bulgaricus cannot grow in synthetic media.     

Efficient free fatty acid production in engineered Escherichia coli strains using soybean oligosaccharides as feedstock

To be competitive with current petrochemicals, microbial synthesis of free fatty acids can be made to rely on a variety of renewable resources rather than on food carbon sources, which increase its attraction for governments and companies. Industrial waste soybean meal is an inexpensive feedstock, which contains soluble sugars such as stachyose, raffinose, sucrose, glucose, galactose, and fructose. Free fatty acids were produced in this report by introducing an acyl‐ACP carrier protein thioesterase and (3R)‐hydroxyacyl‐ACP dehydratase into E. coli. Plasmid pRU600 bearing genes involved in raffinose and sucrose metabolism was also transformed into engineered E. coli strains, which allowed more efficient utilization of these two kinds of specific oligosaccharide present in the soybean meal extract. Strain ML103 (pRU600, pXZ18Z) produced ～1.60 and 2.66 g/L of free fatty acids on sucrose and raffinose, respectively. A higher level of 2.92 g/L fatty acids was obtained on sugar mixture. The fatty acid production using hydrolysate obtained from acid or enzyme based hydrolysis was evaluated. Engineered strains just produced ～0.21 g/L of free fatty acids with soybean meal acid hydrolysate. However, a fatty acid production of 2.61 g/L with a high yield of 0.19 g/g total sugar was observed on an enzymatic hydrolysate. The results suggest that complex mixtures of oligosaccharides derived from soybean meal can serve as viable feedstock to produce free fatty acids. Enzymatic hydrolysis acts as a much more efficient treatment than acid hydrolysis to facilitate the transformation of industrial waste from soybean processing to high value added chemicals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:686–694, 2015     

The production of a new tempeh-like fermented soybean containing a high level of gamma-aminobutyric acid by anaerobic incubation with Rhizopus

A cultivation procedure for the preparation of a new tempeh-like fermented soybean containing a high level of gamma-aminobutyric acid was developed. Steamed soybeans were incubated aerobically with Rhizopus microsporus var. oligosporus IFO 8631 for 20 h, and then anaerobically incubated for 5 h by replacement of the atmosphere with nitrogen. The GABA content in the aerobically fermented soybeans was about 30 mg per 100 g dry fermented soybeans, while the anaerobically cultivation was about 370 mg/100 g dry fermented soybeans. The incubation with several strains of Rhizopus species showed that all of R. microsporus var. oligosporus and R. oryzae examined accumulated GABA in the anaerobically fermented soybeans. In particular, R. microsporus var. oligosporus IFO 32002 and IFO 32003 showed the highest content of GABA (1,740 mg/100 g dry fermented soybeans and 1,500 mg/100 g dry fermented soybeans, respectively). Moreover, the free protein amino acids increased greatly in the fermented soybeans during the anaerobic cultivation.     

Free amino acid content of phloem sap from Brassica plants in relation to performance of Lipaphis erysimi (Hemiptera: Aphididae)

Cultivars of Brassica juncea, B. napus and B. campestris, differing in host-suitability to the mustard aphid (Lipaphis erysimi pseudobrassicae Kalt.), were analysed for their phloem sap content of free amino acids. Sap was collected from excised mustard aphid stylets and analysed by means of high performance liquid chromatography. Aspartic and glutamic acid were, together with glutamine, the dominating amino acids. All plants had very little glycine, γ-aminobutyric acid (GABA), methionine and tryptophan in their sap. This amino acid pattern closely resembled that of cereals. The total concentration and the individual composition of amino acids were not found to be related to the level of aphid resistance previously found in the investigated Brassica species. However, one B. campestris cultivar, yellow sarson YSB-9, on which the aphids were shown to grow more slowly, had less amino acid content than other B. campestris varieties. The slow population growth of aphids on B. juncea, compared to that on B. campestris, is probably due to other than nutritional factors.     

Effect of α-Amino-4-Phosphonobutyrate on the Release of Endogenous Glutamate and Aspartate from Cortical Synaptosomes of Epileptic Rats

The effect of the glutamate antagonist alpha-amino-4-phosphonobutyrate (APBA) on the release of endogenous amino acids from sensorimotor cortical synaptosomes of rats with a cortical cobalt focus and from non-epileptic rats was studied: (1) The release of endogenous glutamate, aspartate, and gamma-aminobutyric acid (GABA) from synaptosomal preparations of cobalt-induced epileptogenic tissues was increased compared with the release from the contralateral (sensorimotor) region or the sensorimotor cortex of normal animals. The intrasynaptosomal content of these amino acids was reduced in proportion to the amount released. The levels of other amino acids were unaffected or showed much smaller changes. (2) APBA (0.5-1 mM) decreased significantly the spontaneous release of aspartate and glutamate from the epileptic foci without affecting GABA or any other amino acid. (3) APBA produced no effect whatsoever on the release of any amino acid from synaptosomal preparations of nonepileptic focus.     

Influence of pumpkin seed cake and extruded linseed on milk production and milk fatty acid profile in Alpine goats

The aim was to determine the effect of substituting pumpkin seed cake (PSC) or extruded linseed (ELS) for soya bean meal in goats’ diets on milk yield, milk composition and fatty acids profile of milk fat. In total, 28 dairy goats were divided into three groups. They were fed with concentrate mixtures containing soya bean meal (Control; n=9), ELS (n=10) or PSC (n=9) as main protein sources in the trial lasting 75 days. Addition of ELS or PSC did not influence milk yield and milk gross composition in contrast to fatty acid profile compared with Control. Supplementation of ELS resulted in greater branched-chain fatty acids (BCFA) and total n-3 fatty acids compared with Control and PSC (P<0.05). Total n-3 fatty acids were accompanied by increased α-linolenic acid (ALA, C18:3n-3; 0.56 g/100 g fatty acids) and EPA (C20:5n-3; 0.12 g/100 g fatty acids) proportions in milk of the ELS group. In contrast, ELS and PSC resulted in lower linoleic acid (LA, C18:2n-6; 2.10 and 2.28 g/100 g fatty acids, respectively) proportions compared with Control (2.80 g/100 g fatty acids; P<0.05). Abovementioned resulted in lower LA/ALA ratio (3.81 v. 7.44 or 6.92, respectively; P<0.05) with supplementation of ELS compared with Control or PSC. The PSC diet decreased total n-6 fatty acids compared with the Control (2.96 v. 3.54 g/100 g fatty acids, P<0.05). Oleic acid (c9-C18:1), CLA (c9,t11-18:2) and t10-,t11-C18:1 did not differ between treatments (P⩾0.08), although stearic acid (C18:0) increased in ELS diets compared with Control (12.7 v. 10.2 g/100 g fatty acids, P<0.05). Partially substituted soya bean meal with ELS in hay-based diets may increase beneficial n-3 fatty acids and BCFA accompanied by lowering LA/ALA ratio and increased C18:0. Pumpkin seed cake completely substituted soya bean meal in the diet of dairy goats without any decrease in milk production or sharp changes in fatty acid profile that may have a commercial or a human health relevancy.