δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.     

Kojic acid–amino acid conjugates as tyrosinase inhibitors

Kojic acid (KA), a well known tyrosinase inhibitor, has insufficient inhibitory activity and stability. We modified KA with amino acids and screened their tyrosinase inhibitory activity. Among them, kojic acid–phenylalanine amide (KA-F-NH₂) showed the strongest inhibitory activity, which was maintained for over 3 months at 50 °C, and acted as a noncompetitive inhibitor as determined by kinetic analysis. It also exhibited dopachrome reducing activity. We also propose a new tyrosinase inhibition mechanism based on the docking simulation data.     

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.     

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.     

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.     

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.     

Production of ω-hydroxyundec-9-enoic acid and n-heptanoic acid from ricinoleic acid by recombinant Escherichia coli-based biocatalyst

ω-Hydroxyundec-9-enoic acid and n-heptanoic acid are valuable building blocks for the production of flavors and antifungal agents as well as bioplastics such as polyamides and polyesters. However, a biosynthetic process to allow high productivity and product yield has not been reported. In the present study, we engineered an Escherichia coli-based biocatalytic process to efficiently produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid from a renewable fatty acid (i.e., ricinoleic acid). Expression systems for catalytic enzymes (i.e., an alcohol dehydrogenase of Micrococcus luteus, a Baeyer–Villiger monooxygenase of Pseudomonas putida KT2440, an esterase of Pseudomonas fluorescens SIK WI) and biotransformation conditions were investigated. Biotransformation during stationary growth phase of recombinant E. coli in a bioreactor allowed to produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid at a rate of 3.2 mM/h resulting in a final product concentration of ca. 20 mM. The total amount of ω-hydroxyundec-9-enoic acid and n-heptanoic acid produced reached 6.5 g/L (4.0 g/L of ω-hydroxyundec-9-enoic acid and 2.5 g/L of n-heptanoic acid). These results indicate that the high value carboxylic acids ω-hydroxyundec-9-enoic acid and n-heptanoic acid can be produced from a renewable fatty acid via whole-cell biotransformation.     

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.     

Eicosapentaenoic acid and docosahexaenoic acid reduce interleukin-1β-mediated cartilage degradation

Introduction  

In inflammatory joint disease, such as osteoarthritis (OA), there is an increased level of proinflammatory cytokines, such as interleukin (IL)-1β. These cytokines stimulate the production of matrix metalloproteinases (MMPs), which leads to the degradation of the cartilage extracellular matrix and the loss of key structural components such as sulphated glycosaminoglycan (sGAG) and collagen II. The aim of this study was to examine the therapeutic potential of n-3 polyunsaturated fatty acids (PUFAs) in an in vitro model of cartilage inflammation.     

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.     

β-hydroxypropionic acid production by Byssochlamys sp. grown on acrylic acid

A strain of Byssochlamys sp. produced -hydroxypropionic acid when grown on media containing high concentrations of acrylic acid. The maximal production of -hydroxypropionic acid was 4.8% when the initial culture medium contained 7% acrylic acid and 2% glucose, and the initial culture pH was adjusted to 7.0. -Hydroxypropionic acid production from acrylic acid depended greatly on the pH of the culture medium. Calcium hydroxide was the best neutralizer. Correspondence to: K. Takamizawa     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Is docosahexaenoic acid more effective than eicosapentaenoic acid for increasing calcium bioavailability?

Experimental animal and human studies have indicated that long chain polyunsaturated fatty acids (LCPUFA) may enhance calcium absorption, reduce urinary calcium excretion, and increase bone calcium content. In the present study, the effect of LCPUFA, as provided in evening primrose oil, fish and tuna oils, on calcium bioavailability was investigated. Growing male rats were fed a semi-synthetic diet for 6 weeks, after which calcium absorption, bone mineral density (ex vivo), bone calcium content, and bone biomechanics were measured. Calcium absorption, ex vivo bone mineral density, and bone calcium content were significantly higher in the animals fed tuna oil compared with those of a control group fed corn oil. Significant correlations were found between the docosahexaenoic acid (DHA) (22:6n-3) content of the red cell membranes and bone density and bone calcium content. DHA increased accretion of calcium in bone significantly more so than eicosapentaenoic acid (EPA) (20:5n-3).     

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.     

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.     

What makes the bioactive lipids phosphatidic acid and lysophosphatidic acid so special?

Phosphatidic acid and lysophosphatidic acid are minor but important anionic bioactive lipids involved in a number of key cellular processes, yet these molecules have a simple phosphate headgroup. To find out what is so special about these lipids, we determined the ionization behavior of phosphatidic acid (PA) and lysophosphatidic acid (LPA) in extended (flat) mixed lipid bilayers using magic angle spinning 31P NMR. Our data show two surprising results. First, despite identical phosphomonoester headgroups, LPA carries more negative charge than PA when present in a phosphatidylcholine bilayer. Dehydroxy-LPA [1-oleoyl-3-(phosphoryl)propanediol] behaves in a manner identical to that of PA, indicating that the difference in negative charge between LPA and PA is caused by the hydroxyl on the glycerol backbone of LPA and its interaction with the phosphomonoester headgroup. Second, deprotonation of phosphatidic acid and lysophosphatidic acid was found to be strongly stimulated by the inclusion of phosphatidylethanolamine in the bilayer, indicating that lipid headgroup charge depends on local lipid composition and will vary between the different subcellular locations of (L)PA. Our findings can be understood in terms of a hydrogen bond formed within the phosphomonoester headgroup of (L)PA and its destabilization by competing intra- or intermolecular hydrogen bonds. We propose that this hydrogen bonding property of (L)PA is involved in the various cellular functions of these lipids.     

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.