Effect of Nitrogen Limitation on the Growth and Lipid Composition of the Green Alga <Emphasis Type="Italic">Botryococcus braunii</Emphasis> Kutz IPPAS H-252

The effect of nitrogen limitation in a medium on the composition of intracellular lipids in the alga Botryococcus braunii Kutz IPPAS H-252 in the course of culture development was investigated. Under the conditions of nitrogen limitation, the alga under investigation accumulated lipids as triacylglycerols, and this process was accompanied by substantial changes in the total fatty acid (FA) composition, which were manifested in a decrease in trienoic acids (from 52.8–57.2 to 19.5–24.7% of total FAs) and an increase in the content of oleic (from 1.1–1.2 to 17.1–24.4%) and saturated (from 23.7–26.0 to 32.9–46.1%) acids. In the control culture, the directionality of FA redistribution was less marked, and these changes were noticed at the later stages of culture development. Under nitrogen limitation, marked changes in the FA composition of polar lipids occurred by the 13th day, and they were characterized by an increase in the content of saturated acids (up to 76.8%) and a dramatic decrease in the content of all polyenoic acids (up to 6.8%). The changes in the FA composition of triacylglycerols were noticed as early as by the 7th day; these changes consisted in an increase in the content of oleic acid, and its high content (28.4–38.4%) was maintained up to the end of culturing. In the control culture, triacylglycerols with a high content of oleic acid were found by the 13th day, although, by this time, the content of total lipids and triacylglycerols did not change.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 357–365.Original Russian Text Copyright © 2005 by Zhila, Kalacheva, Volova.     

The influence of phenylalanine on accumulation of rosmarinic and caffeic acids by Lavandula vera MM cell culture

Lavandula vera MM cell suspension culture was grown in Linsmayer-Skoog medium with different concentrations of phenylalanine. Adding phenylalanine to the medium (0.1–0.5 g/l) enhanced accumulation of caffeic acid in parallel with rosmarinic acid. When 0.3 g phenylalanine/l was added, the yield of rosmarinic and caffeic acids reached 87 mg/l and 60 mg/l respectively, compared with 68 mg/l and 4 mg/l in controls (without phenylalanine).     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

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

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

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l^–1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l^–1.     

Survival of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> during heating,drying and storage in the dried state when growth has occurred in the presence of sucrose or monosodium glutamate

Spray-dried cells of Lactobacillus sakei CTC 494 survived ca. 60% longer in the spray dried state when cells were grown in the presence of 20g sucrose l^–1or 12.5g monosodium glutamate l^–1. No significant differences were observed in viability during storage in the freeze dried state with the addition of these compounds to the growth medium, nor in survival during a heat treatment (55°C). Both sucrose and glutamate in the growth medium suppressed intracellular accumulation of total amino acids and changed the overall pattern of the individual amino acids. Glutamate in the growth medium enhanced intracellular glutamate by ca. 38%. Revisions requested 4 November 2004; Revisions received 13 December 2004     

Structural Changes of Cell Surface in Callus of <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench. during Induction of Morphogenesis

Scanning electron microscopy was used to study the structure of callus of Fagopyrum esculentum Moench. during induction of morphogenesis. It was found that transferring of callus on the medium lacking growth regulators induced several simultaneous developmental programs: embryoidogenesis, rhizogenesis, and gemmogenesis. The program of embryoidogenesis prevailed over the others. Various morphological structures were found to appear on the callus surface in the following order: trichome (on the 2nd day), roots (the 2nd or 3rd day), embryoids (the 6th to 8th day), and buds (the 10th to 14th day). It was shown that morphogenesis is preceded by loosening of the callus surface and by appearance of the secret on it. Possible functions of secretion in the induction of the observed processes are discussed.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 430–437.Original Russian Text Copyright © 2005 by Rumyantseva, Salnikov, Lebedeva.     

The effect of some amino acids on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum Thom 1910

The effect of some amino acids, added to the medium either during inoculation or in the stationary growth phase, on the growth and biosynthesis of ergot alkaloids and quinocitrinins in the fungus Penicillium citrinum VKM FW-800 has been studied. Exogenously added amino acids were mostly utilized in primary metabolism. When added during inoculation, tryptophan and leucine virtually did not influence fungal growth and synthesis of the alkaloids, whereas the addition of isoleucine enhanced the biomass accumulation. When added in the stationary growth phase, tryptophan stimulated the synthesis of both ergot alkaloids and quinocitrinins. Leucine added in the stationary growth phase did not influence the synthesis of ergot alkaloids but inhibited the synthesis of quinocitrinins. Isoleucine inhibited the synthesis of both ergot alkaloids and quinocitrinins irrespective of the time of its addition to the medium.     

Time course of the content of folates and free amino acids in leaves of Pisum sativum L. after irradiation with ultraviolet C

The effect of irradiation with UV-C on the time course of the content of total folates and free amino acids in leaves of pea (Pisum sativum L.) cultivar Neistoshchimyi was studied. It was shown that photolysis of folates is a rapid response to exposure to ultraviolet, as a result of which the plant produces a stable compound, pterin-6-carboxylic acid, with a relative fluorescence quantum yield 2.0 at 20°C (total value, 0.58). Presumably, this compound may be involved in the pterin-mediated photosensitization of singlet oxygen production. The kinetics of changes in the composition of free amino acids after exposure to UV-C has been studied. Exposure to UV-C for 0.5 and 1 min induced utilization of free amino acids, suggesting activation of the synthesis of hormones and alkaloids that may facilitate resistance to the stressor. Greater doses as a result of exposure to radiation for 10 and 40 min decreased the content of free hydrophobic amino acids. This phenomenon could be due to the formation of covalent cross-links in membranes, which decrease the accessibility of hydrophobic amino acids. It is concluded that the changes in the qualitative and quantitative composition of free amino acids in leaves of irradiated plants were due to glycolysis.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 155–161.Original Russian Text Copyright © 2005 by Stakhov, Ladygin, Stakhova.     

In situ adsorption for enhanced alkaloid production byCatharanthus roseus

Summary A significant fraction (10–40%) of the indole alkaloids produced byCatharanthus roseus was observed to be secreted into the medium. When a neutral polymeric resin, known to adsorb these alkaloids, was added to the cultivation medium, the accumulation of total indole alkaloids and the specific alkaloids, ajmalicine and serpentine were stimulated. Sorbent addition was also observed to result in increased ratios of ajmalicine to serpentine, which suggests the potential of using in situ adsorption to direct metabolism toward a specific product or intermediate within a given pathway.     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

Effect of Light Quality on Nitrogen Metabolism of Radish Plants

The long-term action of blue or red light on nitrogen metabolism was studied in radish (Raphanus sativus L.) plants. The potential activity of nitrate reductase (NR) in vivo and its maximum activity in vitro, the content of soluble protein and free amino acids were determined in the course of the growth of a third leaf of radish plants. The effect of light quality on NR activity was found to depend significantly on the stage of leaf development. Blue light (BL) stimulated NR activity in leaves, when their areas were about 11–13% of the fully developed leaves. The efficiency of red light (RL) was significantly lower, because the maximum NR activity was observed in the leaves developed to the stage, when their areas were 38–40% of the final one. The comparative analysis of the pool of free amino acids in expanding leaves of BL- or RL-grown plants revealed significant changes in the contents of individual amino acids. Despite a higher accumulation of two amino acids in the leaves of BL-grown plants, namely, Asp (27% as compared to 13–16% in the RL-grown leaves) and Gly (5% against 2.5% in RL-grown leaves), the BL-grown leaves also demonstrated a significant decrease in Ala (10% as compared to 23% in the RL-grown leaves) and some decrease in the amounts of Ser and Gly. The content of soluble protein in a juvenile BL-grown leaf was observed to decrease gradually during leaf development. However, the protein content in the BL-grown leaf was always higher than in the RL-grown leaf of the same age. We concluded that the photoregulatory action of BL on NR activity determined the different rates of nitrogen assimilation in BL- and RL-grown plants.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 349–356.Original Russian Text Copyright © 2005 by Maevskaya, Bukhov.     

Effect of sulfite on compositional changes of cell constituents in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L., which were preincubated at 25 C for three days in the dark, were suspended in 1 mM potassium phosphate buffer, pH 6.0, and incubated for four days under continuous red light in the presence or absence of 3 mM sulfite. At day 0, 2 and 4 of the incubation, contents of cell constituents were determined. Total lipid content decreased continuously over four days of incubation in the absence of sulfite or in the presence of 3 mM sulfate. In contrast, when sulfite was added to the medium, the decrease stopped after day 2. The content of insoluble glucan increased markedly between day 2 and 4 in the medium without sulfite, whereas it decreased continuously for four days in the medium containing sulfite. The protein content decreased promptly by day 2, but its decrease was delayed when 3 mM sulfite was added to the medium. The content of amino acids also decreased by day 2, but it increased thereafter in the absence of sulfite or in the presence of 3 mM sulfate. In the presence of sulfite, however, the content continued to decrease until day 4. The results indicate that 3 mM sulfite in the incubation medium depressed the utilization of reserve lipid and protein, the synthesis of insoluble glucan and the increase of amino acid pool sizes in fern spores.     

Bioconversion of phenyllactate to phenylalanine by bacteria of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

The nature of phenylalanine auxotrophy in Pseudomonas sp. strain M9 was studied. A metabolic defect was found after the aromatic amino acid pathway branches from chorismate, so that the latter could not be converted to phenylpiruvate. It was shown that enzymatic activity of the strain could convert phenyllactate to phenylalanine. The reaction mechanisms were studied, and approaches to increasing the rate of the process and phenylalanine yield were proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 48–52.Original Russian Text Copyright © 2005 by Beloded, Paramonov, Korovina, Mulyashov, Markvichev.     

Amino acids in honeybee worker haemolymph

Summary In honeybee workers proline was the predominant amino acid and comprised from 50% in newly emerged bees up to 80% of total amino acids from the 3^rd day on. The overall concentration averaged at about 20 mM in newly emerging bees, rose to a maximum of about 25mM at the 3^rd–5^th day and decreased in older bees. Essential amino acids decreased by 40% during the first 3 days and thereafter stayed constant. The bulk of amino acids with a lower concentration (from traces to about 2mM) showed either no change in concentration or was higher in newly emerging bees and decreased during the lifespan of the insects. Forager bees, collected after flight, had significantly lower proline concentrations as compared to 22 day old bees collected from the colony, while the concentrations of the bulk of all other amino acids did not change significantly. There was a great variance in the concentration of all amino acids between different colonies but we could not prove dependency on relatedness.     

Novel formation of α-amino acid from α-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of -amino acids and N-acylamino acids from -oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5–39% yield after hydrolysis with 6N HCl. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3–7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from -ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenyl-alanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Effect of feeding precursors on phenylalanine ammonia-lyase activity and coumarin accumulation in leaves of <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.

Four-week-old chamomile (Matricaria chamomilla) plants were exposed for 72 h to 0.01, 0.1 and 1 mM phenylalanine (Phe) or tyrosine (Tyr). Phe at all concentrations significantly increased phenylalanine ammonia-lyase (PAL) activity (by 30, 76 and 90%, respectively) as well as accumulation of coumarin-related compounds (herniarin and its precursors (Z)- and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids). Free Phe content increased significantly at the highest dose tested. Lower Tyr concentrations (0.01 and 0.1 mM) significantly increased PAL activity and increased free Tyr content, however free Phe content decreased. This indicated that Tyr-mediated stimulation of PAL is coupled to Phe consumption. Notwithstanding, Tyr had no effect on coumarin accumulation. Therefore we speculate that in chamomile a regulation/signalling mechanism could be operating in the pathway leading to coumarin synthesis. The malondialdehyde accumulation, an usual marker of stress in plants, was not significantly changed by amino acid supplements, suggesting that membrane damage is not the signal causing coumarin accumulation. In parallel experiment we observed that neither lower (0.25 × full strength), nor higher (3 × full strength) nitrogen concentration of nutrient solution compared to normal (1 × full strength, 205 mg N l^-1) solution used for Phe/Tyr supply affected herniarin and GMCAs accumulation. This indicates that Phe had stimulatory effect on PAL activity and coumarin metabolism.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine