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.     

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.     

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.     

Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway

Biotin synthesis requires the C7 α,ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.     

Influence of organic supplements on accumulation of β-boswellic acid, acetyl-β-boswellic acid, 11-keto-β-boswellic acid and acetyl-11-keto-β-boswellic acid in callus culture of Boswellia serrata Roxb.

Boswellia serrata Roxb. is a source of several bioactive triterpenoids. Boswellic acid, obtained from oleo-gum resin of the tree, is a major potentially bioactive and medicinal compound. Unrestricted exploitation of its natural resource has led to its listing among the threatened and endangered species. Accumulation of the compound through tissue culture seems a promising option. The present work was conducted to study the effect of sodium pyruvate, l-phenylalanine, glycine, ferulic acid and sucrose on the growth of callus and accumulation of four principal isomers of boswellic acids, viz. β-boswellic acid (BBA), acetyl-β-boswellic acid (ABBA), 11-keto-β-boswellic acid (KBBA) and acetyl-11-keto-β-boswellic acid (AKBBA). Callus cultures obtained from embryo explants of Boswellia serrata on Murashige and Skoog medium containing 2.5 μM 6-benzyladenine, 15 μM indole acetic acid and 200 mg l^?1 polyvinyl pyrrolidone was supplemented with varying concentrations of the supplements. Sodium pyruvate was most beneficial for the production of AKBBA (77 folds), BBA (27 folds) and ABBA (27 folds) at 10 mg l^?1 and for KBBA (47 folds) at 5 mg l^?1 when compared with control. It was closely followed by sucrose (50 g l^?1) resulting in KBBA (22-fold), AKBBA (25-fold), BBA (17-fold) and ABBA (10-fold). Glycine, l-phenylalanine and ferulic acid were relatively less effective. It can be concluded that callus cultures manipulated with different concentrations of organic supplements, sodium pyruvate or sucrose, in particular, could be considered as an alternate strategy for direct production of boswellic acid and help in the conservation of the species.     

1β,7β-Dihydroxydehydroabietic acid,a new biotransformation product of dehydroabietic acid by Aspergillus niger

Microbial transformation of dehydroabietic acid by Aspergillus niger afforded the new derivative 1β,7β-dihydroxydehydroabietic acid and the known 1β-hydroxy and 7β-hydroxy derivatives. The structures were elucidated by spectroscopic methods. The compounds were assessed towards Gram (+) and Gram (−) bacteria and showed a weak antimicrobial effect. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid,citric acid,and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme. Correspondence to: M. Kunioka     

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.     

Effect of conjugated linoleic acid, μ-calpain inhibitor,on pathogenesis of Alzheimer's disease

μ-Calpain is a calcium-dependent cysteine protease, which is activated by μM concentration of calcium in vitro. Disrupted intracellular calcium homeostasis leads to hyper-activation of μ-calpain. Hyper-activated μ-calpain enhances the accumulation of β-amyloid peptide by increasing the expression level of β-secretase (BACE1) and induces hyper-phosphorylation of tau along with the formation of neurofibrillary tangle by mediating p35 cleavage into p25, both of which are the major mechanisms of neurodegeneration in Alzheimer's disease (AD). Hence, inhibition of μ-calpain activity is very important in the treatment and prevention of AD. In this study, conjugated linoleic acid (CLA), an eighteen-carbon unsaturated fatty acid, was discovered as a μ-calpain-specific inhibitor. CLA showed neuroprotective effects against neurotoxins such as H₂O₂ and Aβ_1–42 in SH-SY5Y cells, and inhibited Aβ oligomerization/fibrillation and Aβ-induced Zona Occludens-1 degradation. In addition, CLA decreased the levels of proapoptotic proteins, p35 conversion to p25 and tau phosphorylation. These findings implicate CLA as a new core structure for selective μ-calpain inhibitors with neuroprotective effects. CLA should be further evaluated for its potential use as an AD therapeutic agent.     

Corynebacterium glutamicum,a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro‐aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild‐type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L^?1) corresponded to the lower k_La value of 5 h^?1. Above 5 h^?1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L^?1. Taking into account this threshold, a fed‐batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild‐type strain was able to produce 93.6 g L^?1 of succinate, which is one of the highest concentration reported in the literature.     

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.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

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.     

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.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

Transformation of lithocholic acid to a new bile acid, 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella blakesleeana ST-22

Summary A fungus identified as Cunninghamella blakesleeana (Lendner) can carry out 15-hydroxylation of lithocholic acid to a new bile acid (3,15-dihydroxy-5-cholanic acid). By optimizing the fermentation conditions, the amount of the product increased from 0.17 g/l to 1.2 g/l. Hydrophilicity measurements and in vitro cholesterol solubilization tests showed that 3, 15-dihydroxy-5-cholanic acid was as effective as ursodeoxycholic acid in cholesterol solubilization.Abbreviations LCA lithocholic acid (3-hydroxy-5-cholanic acid) - 3, 15-DHC (3, 15-dihydroxy-5-cholanic acid) - DMSO dimethyl sulfoxide - CHES 2-[N-cyclohexylamino]ethanesulfonic acid     

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.     

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.     

Effect of taurocholate on the conversion of 3α, 7α, 12α-Trihydroxy-5β-Cholestan-26-OIC acid into cholic acid

To determine if the conversion of the intermediate, 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oic acid (THCA), into cholic acid is influenced by taurocholate, two rats were infused intravenously with [³H] THCA until they reached a steady state. Taurocholate was then added and infused at a rate of 1 μmole/min/rat for 48 hours. The percentage of [³H] THCA recovered in the bile did not increase indicating that taurocholate does not suppress the conversion of THCA into cholic acid.