Transgenic manipulation of the metabolism of polyamines in poplar cells

The metabolism of polyamines (putrescine, spermidine, and spermine) has become the target of genetic manipulation because of their significance in plant development and possibly stress tolerance. We studied the polyamine metabolism in non-transgenic (NT) and transgenic cells of poplar (Populus nigra x maximowiczii) expressing a mouse Orn decarboxylase (odc) cDNA. The transgenic cells showed elevated levels of mouse ODC enzyme activity, severalfold higher amounts of putrescine, a small increase in spermidine, and a small reduction in spermine as compared with NT cells. The conversion of labeled ornithine (Orn) into putrescine was significantly higher in the transgenic than the NT cells. Whereas exogenously supplied Orn caused an increase in cellular putrescine in both cell lines, arginine at high concentrations was inhibitory to putrescine accumulation. The addition of urea and glutamine had no effect on polyamines in either of the cell lines. Inhibition of glutamine synthetase by methionine sulfoximine led to a substantial reduction in putrescine and spermidine in both cell lines. The results show that: (a) Transgenic expression of a heterologous odc gene can be used to modulate putrescine metabolism in plant cells, (b) accumulation of putrescine in high amounts does not affect the native arginine decarboxylase activity, (c) Orn biosynthesis occurs primarily from glutamine/glutamate and not from catabolic breakdown of arginine, (d) Orn biosynthesis may become a limiting factor for putrescine production in the odc transgenic cells, and (e) assimilation of nitrogen into glutamine keeps pace with an increased demand for its use for putrescine production.     

Genetic manipulation of polyamine metabolism in poplar II: effects on ethylene biosynthesis

Possible competition between polyamine and ethylene metabolisms was studied in two types of transgenic poplar (Populus nigra × maximowiczii) cells: (a) constitutively expressing a mouse ornithine decarboxylase (ODC, EC 4.1.1.17) cDNA under the control of double 35S cauliflower mosaic virus (CaMV) promoter (cell line 2E), and (b) constitutively expressing a Datura S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA under the control of a single 35S CaMV promoter (line PS-18). The 2E cells contained significantly higher putrescine (Put) as well as spermidine (Spd) contents than the non-transgenic (NT) cells. The PS-18 cells contained three- to five-fold lower amounts of Put than the NT cells; their Spd content was either comparable to NT cells (at 3 d of culture) or it was higher than the NT cells (at 6 d of culture). The production of ethylene in the 2E cells was generally higher than in the NT cells throughout the 7-d culture period. Ethylene production in the PS-18 cells was comparable to NT cells. The cellular content of 1-aminocyclo-propane-1-carboxylic acid in the NT and 2E cells was quite similar, while it was slightly lower in the PS-18 cells. It is concluded that in poplar cells the cellular pool of S-adenosylmethionine is probably large enough to satisfy the demand for both polyamine and ethylene production and no competition between the two pathways is apparent.     

Characterization of the polyamine transport system in mouse neuroblastoma cells. Effects of sodium and system A amino acids

The biochemical properties of polyamine transport system have been studied in detail in NB-15 mouse neuroblastoma cells in culture by measuring the uptake of [14C]putrescine under various experimentally imposed pharmacological conditions. Putrescine uptake in the NB-15 mouse neuroblastoma cells appeared to be a sodium-dependent process. Iso-osmotic displacement of Na+ in the assay medium with either choline or Li+ resulted in a linear decrease of putrescine uptake. Gramicidin, a channel-former ionophore, inhibited putrescine uptake by more than 90% at 20 nM. N-Ethylmaleimide at 5 mM or p-chloromercuribenzene sulfonate at 50 microM completely abolished putrescine uptake. Conversely, oxidized glutathione at 10 mM or 5,5'-dithiobis-(2-nitrobenzoic acid) at 5 microM gave a 1.3-1.4-fold stimulation after a 1-h incubation. This polyamine transport system appeared to be subjected to adaptive regulation. Polyamine antimetabolites such as alpha-difluoromethyl ornithine stimulated putrescine uptake whereas preloading of cells with polyamines inhibited putrescine uptake. Preloading cells with neutral amino acids that belong to sodium-dependent transport System A stimulated putrescine uptake by more than 8-10-fold. These results suggested that the polyamine transport system in NB-15 mouse neuroblastoma cells was sodium dependent and shared some characteristics common to other known sodium-dependent transport systems. These characteristics included (a) sensitivity to ionophores, (b) sensitivity to sulfhydryl reagents, and (c) sensitivity to intracellular contents of substrate molecules. Our data also indicated that polyamine transport may be regulated by transport System A amino acids.     

Genetic manipulation of polyamine catabolism in rodents

Activation of polyamine catabolism through the overexpression of spermidine/spermine N1-acetyltransferase (SSAT) in transgenic rodents does not only lead to distorted tissue polyamine homeostasis, manifested as striking accumulation of putrescine, appearance N1-acetylspermidine and reduction of tissue spermidine and/or spermine pools, but likewise creates striking phenotypic changes. The latter include loss of hair, lipoatrophy and female infertility. Forced expression of SSAT modulates skin, prostate and intestinal carcinogenesis, induces acute pancreatitis and blocks early liver regeneration. Although many of these features are directly attributable to altered tissue polyamine pools, some of them are more likely related to the greatly accelerated flux of the polyamines caused by activated catabolism and compensatorily enhanced biosynthesis.     

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.     

Structure of a designed, right-handed coiled-coil tetramer containing all biological amino acids

The previous design of an unprecedented family of two-, three-, and four-helical, right-handed coiled coils utilized nonbiological amino acids to efficiently pack spaces in the oligomer cores. Here we show that a stable, right-handed parallel tetrameric coiled coil, called RH4B, can be designed entirely using biological amino acids. The X-ray crystal structure of RH4B was determined to 1.1 Angstrom resolution using a designed metal binding site to coordinate a single Yb(2+) ion per 33-amino acid polypeptide chain. The resulting experimental phases were particularly accurate, and the experimental electron density map provided an especially clear, unbiased view of the molecule. The RH4B structure closely matched the design, with equivalent core rotamers and an overall root-mean-square deviation for the N-terminal repeat of the tetramer of 0.24 Angstrom. The clarity and resolution of the electron density map, however, revealed alternate rotamers and structural differences between the three sequence repeats in the molecule. These results suggest that the RH4B structure populates an unanticipated variety of structures.     

Engineered systems for the physical manipulation of single cells

Manipulating the physical location of cells is useful both to organize cells in vitro and to separate cells during screening. The quest to manipulate cells on length scales commensurate with their size has led to a host of technologies exploiting optical, chemical, mechanical, electrical, and other phenomena. Researchers interested in organizing cells are gaining the ability to pattern more than two cell types, to create dynamic surfaces, and to pattern cells in the third dimension. In the realm of cell separation for screening, there has been significant progress in miniaturized flow-based optical sorters as well as in sorting following static microscopic observation.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

Genetic manipulation of the metabolism of polyamines in poplar cells. The regulation of putrescine catabolism

We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra x maximowiczii) expressing a mouse (Mus musculus) ornithine (Orn) decarboxylase (odc) cDNA. The transgenic cells produce 3- to 4-fold higher amounts of Put than the NT cells. The rate of loss of Put from the cells and the initial half-life of cellular Put were determined by feeding the cells with [U-(14)C]Orn and [1,4-(14)C]Put as precursors and following the loss of [(14)C]Put in the cells at various times after transfer to label-free medium. The amount of Put converted into spermidine as well as the loss of Put per gram fresh weight were significantly higher in the transgenic cells than the NT cells. The initial half-life of exogenously supplied [(14)C]Put was not significantly different in the two cell lines. The activity of diamine oxidase, the major enzyme involved in Put catabolism, was comparable in the two cell lines even though the Put content of the transgenic cells was severalfold higher than the NT cells. It is concluded that in poplar cells: (a) exogenously supplied Orn enters the cells and is rapidly converted into Put, (b) the rate of Put catabolism is proportional to the rate of its biosynthesis, and (c) the increased Put degradation occurs without significant changes in the activity of diamine oxidase.     

Effect of urea and amino acids of the ornithine cycle on the biomass accumulation and amino acids synthesis by Candida guilliermondii]

When assimilating urea, arginine, ornithine and citrulline as the sole source of nitrogen, C. guilliermondii shows a higher economic coefficient of biomass accumulation (54.2, 59.7, 40.6% respectively) as compared with ammonium sulphate whose coefficient is 35.6%. Nitrogen sources exert a significant influence on the content of essential amino acids in the alcohol soluble fraction of cell biomass. For instance, urea and arginine are responsible for the accumulation of ornithine (220 and 480 mug/100 mg abs. dry weight), arginine (470 and 587 mug), aspartic acid (220 mug), glutamic acid (520 and 444 mug), alanine (460 and 500 mug), whereas ammonium sulphate provides an accumulation of serine--52 mug, glycine--57 mug, gamma-aminobutyric acid--480 mug, phenyl alanine--96 mug and leucine--96 mug.     

Reversed transport of amino acids in Ehrlich cells

Gramicidin induces a marked Na⁺-dependent efflux of amino acids from Ehrlich cells. In absence of Na⁺, gramicidin does not alter the efflux. In presence of gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl₂ is neither Na⁺ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na⁺-dependent route, nor efflux of several other solutes which are transported by Na⁺-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of the operation of the Na⁺-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na⁺ concentration and a reduction of the electrochemical potential difference for Na⁺.     

Suspension density and accumulation ratio of sugars and amino acids in yeasts

Suspension density has a pronounced effect on the transport parameters of monosaccharides, disaccharides and amino acids in all ycast species tested. InLodderomyces elongisporus, the accumulation ratio of 6-deoxy-D-glucose, a nonmetabolized sugar, was as high as 560: 1 at 0.5 mg dry mass per mL but only 10: 1 at 50 mg dry mass per mL. In the low-density range, the temperature optimum was very pronounced (at about 40 °C) and the pH optimum was very clear at pH 4.6. Iodoacetamide (0.5 mmol/L), 2,4-dinitrophenol (0.5 mmol/L), uranyl ions (0.5 mmol/L) and 2-deoxy-D-glucose (10 mmol/L) depressed the accumulation in the low-density range by 42, 97, 96 and 98 %, respectively. Preincubation with 1% sucrose and 1% L-fructose stimulated subsequent accumulation by 40 and 105%, respectively. In the high-density range, there was a poorly pronounced temperature optimum, no pH optimum and little effects of inhibitors except 2,4-dinitrophenol and 2-deoxy-D-glucose which inhibited by 68 and 89% respectively. No stimulation by preincubation with sugars was observed. There was a difference of 0.3 pH units in the intracellular pH of high-density and low-density cells and the membrane potential was -31 mV and -78 mV, respectively, which could not account for the differences in accumulation. However, there was a fine correlation between this accumulation ratio and the activity of the plasma membrane H⁺ATPase.     

Intracellular accumulation of amino acids increases synaptic potentials in rat hippocampal slices

Amino Acids - The application of high concentrations of taurine induces long-lasting potentiation of synaptic responses and axon excitability. This phenomenon seems to require the contribution of a...