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.     

Mechanisms of mechanosensing — mechanosensitive channels,function and re-engineering

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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.     

Multiplicity of brain cysteine sulfinic acid decarboxylase — Purification,characterization and subunit structure

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn²⁺, methione, and other sulfur-containing amino acids and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5–20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 ± 2 kDa subunits. CSAD appears to require Mn²⁺ for its maximum activity. Other divalent cations fail to replace Mn²⁺ in activation of CSAD activity. However, the precise role of Mn²⁺ in the action of CSAD remains to be determined.     

Phosphonic acid analogue of forfenicine — synthesis, antibacterial activity and degradation byPseudomonas fluorescens

Summary Phosphonic acid analogue of forfenicine, amino (p-formylbenzyl)-phosphonic acid, was synthesized and evaluated as antibacterial agent. As indicated by disc diffusion test this compound was found to inhibit significantly the growth ofBacillus subtilis andStaphylococcus aureus and moderately the growth ofEscherichia coli. Resistance ofPseudomonas fluorescens to the action of the aminophosphonate may result from the ability of the strain to degrade this compound.     

Lipoic acid and diabetes—Part III: Metabolic role of acetyl dihydrolipoic acid

Rat liver lipoyl transacetylase catalyzes the formation of acetyl dihydrolipoic acid from acetyl coenzyme A and dihydrolipoic acid. In an earlier paper the formation of acetyl dihydrolipoic from pyruvate and dihydrolipoic acid catalyzed by pyruvate dehydrogenase has been reported. Acetyl dihydrolipoic acid is a substrate for citrate synthase, acetyl coenzyme A carboxylase and fatty acid synthetase. The V_max. for citrate synthase with acetyl dihydrolipoic acid was identical to acetyl coenzyme A (approximately 1 μmol citrate formed/min/mg protein) while the apparent K_m was approximately 4 times higher with acetyl dihydrolipoic acid as the substrate. This may be due to the fact that synthetic acetyl dihydrolipoic acid is a mixture of 4 possible isomers and only one of them may be the substrate for the enzymatic reaction. While dihydrolipoic acid can replace coenzyme A in the activation of succinate catalyzed by succinyl coenzyme A synthetase, the transfer of coenzyme A between succinate and acetoacetyl dihydrolipoic acid catalyzed by succinyl coenzyme A: 3 oxo-acid coenzyme A transferase does not occur.     

Mucor—a source of cocoa butter and gamma-linolenic acid

Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:0) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGs) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SUS-TAGs and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% 18:0 in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGs.M.P. Roux, J.L.F. Kock, A. Botha, J.C. du Preez and P.J. Botes are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa; G.V. Wells is with Sasol Waxes, P.O. Box 1, Sasolburg, South Africa.     

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.     

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.     

Specificity of protein — Nucleic acid interaction and the biochemical evolution

The water soluble carbodiimide mediated condensation of dipeptides of the general form Gly-X was carried out in the presence of mono- and poly-nucleotides. The observed yield of the tetrapeptide was found to be higher for peptide-nucleotide system of higher interaction specificity following mainly the anticodon-amino acid relationship (Basu, H.S. & Podder, S.K., 1981, Ind. J. Biochem. Biophys.,19, 251–253). The yield of the condensation product of L-peptide was more because of its higher interaction specificity. The extent of the racemization during the condensation of Gly-L-Phe, Gly-L-Tyr and Gly-D-Phe was found to be dependent on the specificity of the interaction —the higher the specificity, the lesser the racemization. The product formed was shown to have a catalytic effect on the condensation reaction. These data thus provide a mechanism showing how the specific interaction between amino acids/dipeptides and nucleic acids could lead to the formation of the primitive translation machinery.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

Effect of β-indoleacetic acid,maleic hydrazide,and 2,3,5-triiodobenzoic acid on N,P, K,and Ca accumulation by pea plants

A study was performed on the effect of various concentrations of IAA, 2,3,6-triiodobenzoic acid, and maleic hydrazide, supplied to Richter’s nutrient solution, on growth of pea plants in water cultures. After a 18-day cultivation growth was evaluated and in the plants gathered the content of total N, P, K, and Ca was estimated. Growth of experimental plants (as evaluated from fresh and dry weight) was affected by all three regulators in dependence on the concentration used. It was stimulated by lower concentrations and inhibited by higher, the production of both fresh and dry weight of the root system being stimulated by all IAA concentrations used. The ratio of root dry weight to that of the entire plant was markedly increased after application of IAA and 2,3,5-triiodobenzoic acid, whereas when applying maleic hydrazide it was only slightly increased in comparison with control. Stimulation or inhibition of growth induced by IAA treatment was accompanied by an accordingly increased or decreased accumulation of N, P, K, and Ca. Thus their utilization did not change in comparison with control. On the other hand, both inhibitory and stimulatory effects of 2,3,5-triiodobenzoic acid and maleic hydrazide on growth were associated with a relatively lower accumulation of the elements in question, resulting in an increased utilization. The distribution index of N, P, K, and Ca decreased with increasing concentrations of IAA, 2,3,5-triiodobenzoic acid and maleic hydrazide. Only the highest 2,3,5-triiodobenzoic acid and maleic hydrazide concentrations used brought about a more marked increase in the distribution index of potassium, simultaneously with a marked decrease in the distribution index of calcium.     

The interaction of D-galactonic acid with CrVI and CrIII. Structure,stability and physical properties of CrIII—aldonate complexes

The redox reaction between D-galactonic acid and potassium chromate yields ((lyxonateH₋₁)(galactonateH₋₁)Cr(OH₂))K·H₂O_n, with both aldonate molecules acting as bidentate ligands with the carboxylate and one alkoxo function as the donor sites. The shift of the CO₂^{poststaggered−} stretching vibration towards lower frequencies upon coordination and the high value of Δv indicate that the carboxylate acts as a monodentate donor site. Magnetic susceptibility data for the compound in the temperature range 3–300 K exhibit a drop in the effective magnetic moment with temperature below 70 K, which is indicative of antiferromagnetic interactions between the Cr^III centres. The molar magnetic susceptibility versus temperature plot could be fitted with the Fisher Hamiltonian for the case of infinite chains, equation-modified for the presence of monomeric species. The EPR and UV-Vis spectroscopic studies reveal that, in solution, the complex retains the distorted octahedral local coordination geometry. The ((lyxonateH₋₁)(galactonateH₋₁)Cr(OH₂))K_n dissociates slowly in aqueous solution but faster at high [H⁺], because of the rapid protonation of the alkoxo bridges linking the monomeric units. The potentiometric evaluation of the closely related binary system Cr^III-d-galactonate shows that the (Cr(galactonateH_−n)₂)^{1 − 2n} complexes are the major species in the 4–12 pH range, when a 1:2 Cr^III:ligand ratio is used. ¹³C NMR reveals that theCO₂^{poststaggered−} group is one of the coordination sites of the ligand.     

Can the combination of electrochemical regeneration of NAD+, selectivity of L‐α‐amino‐acid dehydrogenase,and reductive amination of α‐keto‐acid be applied to the inversion of configuration of a L‐α‐amino‐acid?

The inversion of configuration of L‐alanine can be carried out by combining its selective oxidation in the presence of NAD⁺ and L‐alanine dehydrogenase, electrochemical regeneration of the NAD⁺ at a carbon felt anode, and reductive amination of pyruvate, i.e., reduction of its imino derivative at a mercury cathode, the reaction mixture being buffered with concentrated ammonium/ammonia (1.28M / 1.28M). The dehydrogenase exhibits astonishing activity and stability under such extreme conditions of pH and ionic strength. The main drawback of the process is its slowness. At best, the complete inversion of a 10 mM solution of L‐alanine requires 140 h. A careful and detailed quantitative analysis of each of the key steps involved shows that the enzyme catalyzed oxidation is so thermodynamically uphill that it can be driven efficiently to completion only when both the coenzyme regeneration and the pyruvate reduction are very effective. The first condition is easily fulfilled. Under the best conditions, it is the rate of the chemical reaction producing the imine which controls the whole process kinetically. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 101–107, 1999.     

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

Dietary linoleic acid has no effect on arachidonic acid,but increases n-6 eicosadienoic acid,and lowers dihomo-γ-linolenic and eicosapentaenoic acid in plasma of adult men

High intakes of linoleic acid (LA,18:2n-6) have raised concern due to possible increase in arachidonic acid (ARA, 20:4n-6) synthesis, and inhibition of alpha linolenic acid (ALA, 18:3n-3) desaturation to eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In healthy men, 10.5% energy compared to 3.8% energy LA with 1% energy ALA increased plasma phospholipid LA and 20:2n-6, the elongation product of LA, and decreased EPA, with no change in ARA. However, LA was inversely related to ARA at both 10.5% energy and 3.8% energy LA, (r=?0.761, r=?0.817, p<0.001, respectively). A two-fold variability in ARA among individuals was not explained by the dietary LA, ARA, ALA, or fish intake. Our results confirm LA requirements for ARA synthesis is low, <3.8% energy, and they suggest current LA intakes saturate Δ-6 desaturation and adversely affect n-3 fatty acid metabolism. Factors other than n-6 fatty acid intake are important modifiers of plasma ARA.     

Bioorganic synthesis, characterization and antioxidant activity of esters of natural phenolics and α-lipoic acid

Chemo-enzymatic synthesis of six esters of natural phenolics and α-lipoic acid was carried to produce novel compounds with potential bioactivity. The synthetic route was mild, simple, and efficient with satisfactory yields. The synthesized compounds were screened for antioxidant activities. The prepared derivatives exhibited very good antioxidant activities as determined by DPPH radical scavenging assay and inhibition of lipid oxidation in fish oil emulsion system. Among the prepared derivatives, three compounds exhibited radical scavenging activity similar to the reference antioxidants, BHT and alpha-tocopherol in the DPPH radical scavenging assay, where as in fish oil emulsion system, two derivatives showed activity, which was similar to the reference antioxidants.     

Oxidant induced injury of erythrocyte—Role of green tea leaf and ascorbic acid

Oxidant and free radical-generating system were used to promote oxidative damage in erythrocytes. Among the oxidants used, phenylhydrazine represents one of the most investigated intracellular free radical-generating probes, which in the presence of haemoglobin autooxidises and give rise to hydroxyl radical, a marker for cellular damage. Erythrocyte, as a single cell, is a good model to be used for studying the haemolytic mechanism of anaemia. Our present investigations reveal increased lipid peroxidation of erythrocyte using phenylhydrazine as well as other oxygen-generating systems (hydrogen peroxide, iron with hydrogen peroxide). It has further been observed that not only lipid peroxidation, phenylhydrazine causes significant elevation in methemoglobin formation, catalase activity and turbidity, in the above system, which are the typical characteristics of haemolytic anaemia. However, exogenous administration of green tea leaf extract and ascorbic acid as natural antioxidants and free radical scavengers were shown to protect separately increased lipid peroxidation caused by phenylhydrazine, though the degree of protection is more in case of green tea leaf extract than ascorbic acid. Results suggest that oxidative damage in vivo due to haemolytic disease may be checked to some extent by using natural antioxidants. (Mol Cell Biochem 276: 205–210, 2005)     

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.     

Enhanced production of δ-aminolevulinic acid, bilipigments, and antioxidants from tropical algae of India

We studied the enhanced production of high quality biomass, δ-aminolevulinic acid (δ-ALA), bilipigments, and antioxidants from five tropical blue green algae (cyanobacteria) in a full factorial design using free and immobilized cells in batch culture. Production of nutraceuticals was high in spray dried powder prepared from immobilized cell cultures. Nostochopsis lobatus showed superiority over rest of the species with respect to bilipigments, δ-ALA, nutritive value, antioxidant capacity, and ascorbate oxidase (APX) activity. Antioxidative capacity of phycobiliproteins extracted from these cyanobacteria (121.15 μM TE/g, Nostoc verrucosum to 217.62 μM TE/g, Nostochopsis lobatus) was invariably higher than those observed for higher plant sources and substantially increased under immobilized cell culture condition. Antioxidative enzyme, ascorbate oxidase remained stable in dry food preparations with considerably high activity under immobilized cell preparations (APX_max, 3.40 μmol/min/mg chlorophyll). These observations have important connotations in light of upcoming food and nutraceutical industries in the global market. Use of immobilized cells in batch culture could be an effective approach for scaling up production for commercial use.