Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Examination of an equilibrium interpretation of deoxyribonucleic acid–ribonucleic acid hybridization data

1. When a constant amount of denatured DNA is annealed for a constant time with a series of different RNA concentrations, it is often observed that the reciprocal of the amount of RNA hybridized is linearly proportional to the reciprocal of the RNA concentration. This may be explained by assuming that an equilibrium is set up between free RNA and DNA on the one hand and DNA-RNA hybrid on the other. The hybridization of Escherichia coli DNA and ribosomal RNA was used to test this proposition. Rate constants were estimated from the initial rates of the forward and back reactions and compared with direct estimates of the dissociation constant. 2. The rate constants of the forward and back reactions were estimated to be 1.82mlmug(-1)h(-1) (160lmol(-1)s(-1)) and 0.023h(-1) (6.4x10(-6)s(-1)) respectively, giving a ratio k(2)/k(1)=0.013mugml(-1). After 24h annealing the dissociation constant was estimated to be 0.114mugml(-1), and by extrapolation to infinite time, 0.047mugml(-1). 3. It is concluded that (a) equilibrium greatly favours the hybrid complex, (b) equilibrium is not established in 24h, (c) the equilibria that were directly estimated are incompatible either with the measured rates of the forward and back reactions or with the simple formulation of the reaction that was adopted, and finally (d) for these reasons the equilibrium interpretation of the linear reciprocal relationship is unsatisfactory.     

First partial synthesis of α-boswellic acid from oleanolic acid

While β-boswellic acid is very readily available by extraction from frankincense resin, the accessibility of α-boswellic from the resin involved great effort and tedious purification procedures. Alternatively, a partial synthesis from readily available oleanolic acid was developed, the key steps of which are a reduction of the carboxyl group C-28 furnishing a methyl group, followed by palladium-assisted oxidation of the methyl group C-24, and configurational inversion at C-3.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an -lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of -lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, -lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and -ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an -lipoic acid, coenzyme A, and pyruvate or -ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an -lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.     

Unique biosynthesis of dehydroquinic acid?

A search of the genomic sequences of the thermophilic microorganisms Aquifex aeolicus, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii for the first seven enzymes (aroG, B, D, E, K, A, and C ) involved in the shikimic acid biosynthetic pathway reveal two key enzymes are missing. The first enzyme in the pathway, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (aroG) and the second enzyme in the pathway, 5-dehydroquinic acid synthase (aroB) are "missing." The remaining five genes for the shikimate pathway in these organism are present and are similar to the corresponding Escherichia coli genes. The genomic sequences of the thermophiles Pyrococcus abyssi and Thermotoga maritima contain the aroG and aroB genes. Several fungi such as Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pneumocystis carinii f. sp. carinii, and Neurospora crassa contain the gene aroM, a pentafunctional enzyme whose overall activity is equivalent to the combined catalytic activities of proteins expressed by aroB, D, E, K, and A genes. Two of these fungi also lack an aroG gene. A discussion of potential reasons for these missing enzymes is presented.     

The effect of genetic complexity on the time-course of ribonucleic acid–deoxyribonucleic acid hybridization

1. The rate of RNA-DNA hybridization was studied under conditions of RNA excess, with RNA synthesized in vitro. The initial rate of the reaction was proportional to the initial RNA concentration. Throughout the observed course of the reaction there was a linear relationship between the reciprocal of the amount of RNA hybridized/mug. of DNA and the reciprocal of time. The slope of the reciprocal plot was inversely proportional to the initial RNA concentration. 2. A comparison was made of the hybridization of DNA from Escherichia coli and from bacteriophages T4 and lambda with homologous RNA. The initial rate of hybridization was inversely proportional to the genetic complexity of the hybridizing system. The slope of the reciprocal-time plot was directly proportional to genetic complexity. These results are interpreted to indicate that the rate of hybridization reflects the mean concentration of the various unique RNA species in a preparation.     

Non-enzymic β-decarboxylation of aspartic acid

Summary Non-enzymic-decarboxylation of aspartic acid at 85° is catalyzed by Al³⁺ and pyridoxal. The reaction is optimum at pH 4.0. Both Al³⁺ and pyridoxal are specifically required because replacing these by other cations or by other vitamin B₆ derivatives greatly lowers the formation of alanine. Conversion of 8 µmoles of aspartic acid to alanine is optimum in presence of 1µmole of Al³⁺ and 5 µmoles of pyridoxal. Increasing the concentration of pyridoxal to more than 5 µmoles lowers the alanine formation by the latter being converted to pyruvate by transamination with the excess pyridoxal.Studies on the mechanism of decarboxylation suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The results are interpreted to suggest that the non-enzymic aspartate-decarboxylation process is closely related to and inseparable from the non-enzymic transamination process in a manner analogous to that reported for the highly purified asparate-decarboxylase. The possible significance of these results to prebiotic molecular evolution is briefly discussed.     

Effects of capric acid on rumen methanogenesis and biohydrogenation of linoleic and α-linolenic acid

Capric acid (C10:0), a medium chain fatty acid, was evaluated for its anti-methanogenic activity and its potential to modify the rumen biohydrogenation of linoleic (C18:2n-6) and α-linolenic acids (C18:3n-3). A standard dairy concentrate (0.5 g), supplemented with sunflower oil (10 mg) and linseed oil (10 mg) and increasing doses of capric acid (0, 10, 20 and 30 mg), was incubated with mixed rumen contents and buffer (1 : 4 v/v) for 24 h. The methane inhibitory effect of capric acid was more pronounced at the highest (30 mg) dose compared to the medium (20 mg) (-85% v. -34%), whereas the lower dose (10 mg) did not reduce rumen methanogenesis. A 23% decrease in total short-chain fatty acid (SCFA) production was observed, accompanied by shifts towards increased butyrate at 20 mg and increased propionate at 30 mg of capric acid (P < 0.001). Capric acid linearly decreased the extent of biohydrogenation of C18:2n-6 and C18:3n-3, by up to 60% and 86%, respectively. This reduction was partially due to a lower extent of lipolysis when capric acid was supplemented. Capric acid at 20 and 30 mg completely inhibited the production of C18:0 (P < 0.001), resulting in an accumulation of biohydrogenation intermediates, mainly C18:1t10 + t11 and C18:2t11c15. In contrast to effects on rumen fermentation (methane production and proportions of SCFA), 30 mg of capric acid did not induce major changes in rumen biohydrogenation as compared to the medium (20 mg) dose. This study revealed the dual action of capric acid, being inhibitory to both methane production and biohydrogenation of C18:2n-6 and C18:3n-3.     

Structure–activity relationship investigation of tertiary amine derivatives of cinnamic acid as acetylcholinesterase and butyrylcholinesterase inhibitors: compared with that of phenylpropionic acid,sorbic acid and hexanoic acid

In the present investigation, 48 new tertiary amine derivatives of cinnamic acid, phenylpropionic acid, sorbic acid and hexanoic acid (4d–6g, 10d–12g, 16d–18g and 22d–24g) were designed, synthesized and evaluated for the effect on AChE and BChE in vitro. The results revealed that the alteration of aminoalkyl types and substituted positions markedly influences the effects in inhibiting AChE. Almost of all cinnamic acid derivatives had the most potent inhibitory activity than that of other acid derivatives with the same aminoalkyl side chain. Unsaturated bond and benzene ring in cinnamic acid scaffold seems important for the inhibitory activity against AChE. Among them, compound 6g revealed the most potent AChE inhibitory activity (IC₅₀ value: 3.64?µmol/L) and highest selectivity over BChE (ratio: 28.6). Enzyme kinetic study showed that it present a mixed-type inhibition against AChE. The molecular docking study suggested that it can bind with the catalytic site and peripheral site of AChE.     

Characterization of rapidly labelled ribonucleic acid in Escherichia coli by deoxyribonucleic acid–ribonucleic acid hybridization

1. Rapidly labelled RNA from Escherichia coli K 12 was characterized by hybridization to denatured E. coli DNA on cellulose nitrate membrane filters. The experiments were designed to show that, if sufficient denatured DNA is offered in a single challenge, practically all the rapidly labelled RNA will hybridize. With the technique employed, 75-80% hybridization efficiency could be obtained as a maximum. Even if an excess of DNA sites were offered, this value could not be improved upon in any single challenge of rapidly labelled RNA with denatured E. coli DNA. 2. It was confirmed that the hybridization technique can separate the rapidly labelled RNA into two fractions. One of these (30% of the total) was efficiently hybridized with the low DNA/RNA ratio (10:1, w/w) used in tests. The other fraction (70% of the total) was hybridized to DNA at low efficiencies with the DNA/RNA ratio 10:1, and was hybridized progressively more effectively as the amount of denatured DNA was increased. A practical maximum of 80% hybridization of all the rapidly labelled RNA was first achieved at a DNA/RNA ratio 210:1 (+/-10:1). This fraction was fully representative of the rapidly labelled RNA with regard to kind and relative amount of materials hybridized. 3. In competition experiments, where additions were made of unlabelled RNA prepared from E. coli DNA, DNA-dependent RNA polymerase (EC 2.7.7.6) and nucleoside 5'-triphosphates, the rapidly labelled RNA fraction hybridized at a low (10:1) DNA/RNA ratio was shown to be competitive with a product from genes other than those responsible for ribosomal RNA synthesis and thus was presumably messenger RNA. At higher DNA/rapidly labelled RNA ratios (200:1), competition with added unlabelled E. coli ribosomal RNA (without messenger RNA contaminants) lowered the hybridization of the rapidly labelled RNA from its 80% maximum to 23%. This proportion of rapidly labelled RNA was not competitive with E. coli ribosomal RNA even when the latter was in large excess. The ribosomal RNA would also not compete with the 23% rapidly labelled RNA bound to DNA at low DNA/RNA ratios. It was thus demonstrated that the major part of E. coli rapidly labelled RNA (70%) is ribosomal RNA, presumably a precursor to the RNA in mature ribosomes. 4. These studies have shown that, when earlier workers used low DNA/RNA ratios (about 10:1) in the assay of messenger RNA in bacterial rapidly labelled RNA, a reasonable estimate of this fraction was achieved. Criticisms that individual messenger RNA species may be synthesized from single DNA sites in E. coli at rates that lead to low efficiencies of messenger RNA binding at low DNA/RNA ratios are refuted. In accordance with earlier results, estimations of the messenger RNA content of E. coli in both rapidly labelled and randomly labelled RNA show that this fraction is 1.8-1.9% of the total RNA. This shows that, if any messenger RNA of relatively long life exists in E. coli, it does not contribute a measurable weight to that of rapidly labelled messenger RNA.     

Relationship between transport and metabolism of α-naphthaleneacetic acid, β-naphthaleneacetic acid and α-decalylacetic acid in segments of Coleus

Summary Transportand metabolism of -naphthaleneacetic acid -naphthaleneacetic acid, and -decalylacetic acid, all labelled with ¹⁴C in the carboxyl, group, were studied. Only -naphthaleneacetic acid is transported in a polar way. Most of the radioactivity in the tissue is in a low molecular form, either free or as immobilization products. The immobilization of -naphthaleneacetic acid is similar to that of -naphthaleneacetic acid. Immobilization of -decalylacetic acid is typically different. Bioassays showed -naphthaleneacetic acid as the sole biologically active component. It is concluded that stereo requirements necessary for biological activity are also required for polar auxin transport. It is further concluded that the observed specificity of the transport system is not related to the formation of immobilization products.     

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ₂ and PGA₂, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.     

Quantitative analysis and comparative decarboxylation of aminomalonic acid, β-carboxyaspartic acid,and γ-carboxyglutamic acid

Methods for quantitative analysis of the carboxylated amino acids, aminomalonic acid, β-carboxyaspartic acid, and γ-carboxyglutamic acid, are presented. These substances are acid labile and thus can be measured only after alkaline hydrolysis of proteins and peptides. Half-times for decarboxylation in 1 m HCl at 100°C are: aminomalonic acid (1.2 min); β-carboxyaspartic acid (1.7 min); and γ-carboxyglutamic acid (8.6 min). This property is useful for unequivocal identification in complex hydrolysates.     

Effect of propionic acid on citric acid fermentation in an integrated citric acid–methane fermentation process

In this study, an integrated citric acid-methane fermentation process was established to solve the problem of wastewater treatment in citric acid production. Citric acid wastewater was treated through anaerobic digestion and then the anaerobic digestion effluent (ADE) was further treated and recycled for the next batch citric acid fermentation. This process could eliminate wastewater discharge and reduce water resource consumption. Propionic acid was found in the ADE and its concentration continually increased in recycling. Effect of propionic acid on citric acid fermentation was investigated, and results indicated that influence of propionic acid on citric acid fermentation was contributed to the undissociated form. Citric acid fermentation was inhibited when the concentration of propionic acid was above 2, 4, and 6 mM in initial pH 4.0, 4.5 and, 5.0, respectively. However, low concentration of propionic acid could promote isomaltase activity which converted more isomaltose to available sugar, thereby increasing citric acid production. High concentration of propionic acid could influence the vitality of cell and prolong the lag phase, causing large amount of glucose still remaining in medium at the end of fermentation and decreasing citric acid production.     

Microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on l-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid l-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.     

Ab-initio SCF investigation ofγ-aminobutyric acid

Summary The potential energy surface of the neutral form of-aminobutyric acid was investigated by means of ab-initio 4-31 G SCF calculations. Geometries, energies, and selected wave numbers of all 62 symmetry unique local minima are reported. Intramolecular interactions and all reactions, which involve the intramolecular hydrogen bond, are discussed and compared with those present in the homologues-alanine and glycine.Abbreviations GABA -aminobutyric acid - PES potential energy surface - RHF Roothaan-Hartree-Fock - SCF self consistent field     

Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.     

Changes of γ-linolenic acid content inRhizopus arrhizus duringl(+)-lactic acid fermentation

Effects of nitrogen source, temperature and pH onl(+)-lactic acid production and γ-linolenic acid (GLA) accumulation byRhizopus arrhizus were examined. The nitrogen source had a minor effect on lactate synthesis but influenced the total lipid content and the fatty acid composition in fungus. Higher temperature favorably influenced the rate of both lactic acid production and lipid formation in the biomass and caused a decrease in the yields of oligounsaturated fatty acids. At higher temperature and after glucose exhaustion, degradation of lactate increased. A low pH value negatively affected the formation of lipids and lactate synthesis. The highest value of GLA in the lipid (25.5%,W/W) was reached at the end of lactate synthesis, but maximum yields of total lipids were achieved when the cultivation continued in the presence of lactate until polyols were exhausted.