PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.     

Additive and Synergistic Growth-inhibiting Properties of the Canaline-Urea Cycle Amino Acids

Growth studies with Lemna minor revealed the additive and synergistic growth-inhibiting properties of the canaline-urea cycle amino acids. Simultaneous canavanine and canaline treatment caused an additive reduction in frond production. Ureidohomoserine interacted with canaline or canavanine to affect synergistically L. minor growth by enhancing individual canavanine or canaline toxicity and increasing the additive growth reduction caused by canavanine plus canaline. The ornithineurea cycle amino acids effectively counteracted both the additive and synergistic growth-inhibiting properties of the canaline-urea cycle compounds.     

Amino Acid levels in carrot cell suspension culture: no correlation with aspartate kinase isoenzyme levels

Free and protein amino acids were analyzed during the growth cycle of a suspension culture of carrot (Daucus carota L.) in which there is a 10-fold increase in a lysine-sensitive isoenzyme of aspartate kinase during the early part of the growth cycle. There is little change in either total amino acids or in the amino acids derived from aspartate between 3 and 24 days of culture. It is estimated that the demand for net synthesis of aspartate-derived amino acids is decreased as the growth rate declines and that there is no correlation between the amount of extractable lysine-sensitive aspartate kinase and the amounts of amino acids being synthesized.     

Regulation of development in Dictyostelium discoideum: I. Initiation of the growth to development transition by amino acid starvation

Evidence is presented which indicates that amino acid starvation is the specific stimulus initiating the developmental phase of the life cycle of Dictyostelium discoideum: (i) Amoebae were washed free of complex growth medium and placed in buffer supplemented with specific nutrients; amino acids were the only nutrients that specifically inhibited the initiation of development. (ii) A partially defined growth medium allowing selective starvation for amino acids or glucose during growth is described. Amoebae initiated development only when starved for amino acids. Any effect of glucose on the primary control of the initiation of development is an indirect result of its utilization as a source of precursors for endogenously synthesized amino acids.     

Inhibition of yeast Δ1-pyrroline-5-carboxylate dehydrogenase by common amino acids and the regulation of proline catabolism

A yeast glutamate auxotroph (glt_{1 ? 1}), blocked in the tricarboxylic acid cycle at aconitase, is shown to possess catabolic pathways to glutamate from proline, arginine and glutamine, and grows on any of these amino acids in a minimal medium. This mutant does not, however, grow on these amino acids in a medium containing the full complement of common amino acids minus glutamate. The mechanism of this growth failure involves partial inhibition of the catabolic routes to glutamate by more than half the common amino acids. In the case of proline catabolism, this inhibition is localized principally at the enzyme Δ¹-pyrroline-5-carboxylate: NAD(P)⁺ oxidoreductase by in vitro studies. Similar results with this enzyme prepared both from yeast and from beef kidney mitochondria suggest that the inhibition observed may be the basis of a regulatory mechanism of general significance.     

Accumulation of putrescine and related amino acids in potassium deficient Sesamum

Sesamum indicum was grown in complete or potassium deficient nutrient solution and amino acids, amines, nitrogen and potassium were determined weekly in the leaves. The incorporation of l-arginine-[U-¹⁴C] into protein was also followed. The interconversions of the amino acids of the ordithine-urea cycle, and their contribution to the formation of amines, were studied in cell-free extracts and intact leaves using labelled amino acids. As the level of potassium in the leaves decreased, the levels of the amino acids ornithine, citrulline and arginine, and of the amines putrescine, N-carbamylputrescine and agmatine increased. Potassium deficiency also reduced the rate of protein synthesis. Putrescine appears to be formed preferentially from citrulline with N-carbamylputrescine as intermediate.     

Measurement of N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography–tandem quadrupole mass spectrometry

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional ¹⁵N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M + 1 isotopomers of each natural AQC–amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M + 1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of ¹⁵N enrichment was confirmed by measuring a series of ¹⁵N-labeled arginine standards. The positional ¹⁵N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional ¹⁵N enrichment in the plasma of rats fed ¹⁵N-labeled glutamine. These results suggest the utility of LC–MS/MS detection of AQC–amino acids for the measurement of isotopic enrichment in ¹⁵N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a β-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Acides aminés de microsporidies parasites de crustacés décapodes marins

The amino acids of four species of Microsporidia parasitizing crustaceans were investigated: three species — Thelohania maenadis, Ormieresia carcini and Ameson pulvis — are parasites of Carcinus mediterraneus; Inodosporus sp. is a parasite of Palaemon serratus. Seventeen protein amino acids were identified of which aspartic acid, glutamic acid and lysine were quantitatively the most important, being quantitatively similar in all four parasites. The relative amounts of the next most abundant amino acids were found to vary and might serve as a systematic criterion at the genus level. For example, phenylalanine is predominant in Ormieresia and leucine in Inodosporus: serine in Thelohania and glycine in Ameson are also predominant, but to a lesser extent. The free amino acids composition shows little qualitative variation among the four genera, but quantitative differences are found in the composition of Microsporidia parasitizing the same host species; this may reflect variations in the amino acid metabolism of the parasite. The urea cycle in Ormeresia is most remarkable in this connection. The free amino acid level in the parasites was generally found to be in inverse proportion to the level in the host; the amino acids which are found to exist at high levels in the parasites correspond to essential amino acids of the Crustacea. Metabolic and adaptative relations are discussed.     

Introduced fire ants can exclude native ants from critical mutualist-provided resources

After over 30 years of research, it was recently shown that nectar amino acids increase female butterfly fecundity. However, little attention has been paid to the effect of nectar amino acids on male butterfly reproduction. Here, we show that larval food conditions (nitrogen-rich vs. nitrogen-poor host plants) and adult diet quality (nectar with or without amino acids) affected the amount of consumed nectar in Coenonympha pamphilus males. Furthermore, amino acids in the nectar diet of males increased progeny’s larval hatching mass, irrespective of paternal larval reserves. Our study takes the whole reproductive cycle of male butterflies into account, and also considers the role of females in passing male nutrients to offspring, as males’ realized reproduction was examined indirectly via nuptial gifts, by female performance. With this comprehensive approach, we demonstrate for the first time that nectar amino acids can improve male butterfly reproduction, supporting the old postulate that nectar amino acids generally enhance butterfly fitness.     

Storage of nutriments by adult female Glossina morsitans and their transfer to the intra-uterine larva

Administration of U-¹⁴C arginine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine, or valine into the haemolymph of female Glossina morsitans on the first day of the pregnancy cycle was followed by radiometric analysis of the post-parturient larva. Radioactivity in the larva, expressed as a percentage of the administered activity, was low with histidine (0.3%) and arginine (2.3%) but higher with the other six amino acids (8.2% to 16.8%). ¹⁴C incorporation in the larval lipid was extremely low with arginine and histidine, but with the remaining six amino acids lipids showed the most ¹⁴C labelling. Radioactivity was detected in the larval amino acids corresponding to those injected into the female parents. Further radiometric study using labelled leucine showed that during the first 5 days of pregnancy surplus leucine was largely converted to lipids for larval growth. Thereafter, while the rate of leucine-derived ¹⁴C incorporation in the larval lipids declined rapidly that in the larval proteins increased. Implications are that female G. morsitans has a significant capacity to store nutriments derived from bloodmeals ingested during early pregnancy destined for larval development, and that normal growth of the intra-uterine progeny is a function of optimum feeding throughout the pregnancy cycle.     

Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae

The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.     

The pathways of amino acid oxidation during germination of mustard seeds (Sinapis alba)

During the germination of Sinapis alba seed, alanine and some other amino acids were oxidized via the tricarboxylic acid cycle (TCA), although the pentose phosphate pathway (PPP) was also operative and may account for ca 50% of glucose oxidation. The relative operation of the PPP and the TCA cycle was influenced by changes in the concentrations of glutamic acid and glycine.     

Effect of growth substrates on morphology of Nocardia corallina

Cells of Nocardia corallina ATCC 4273 form multiply branched coenocytic mycelia and subsequent fragment to spherical cells when grown on solidified complex media. In liquid shake cultures using complex media the organisms grow into pleomorphic but seldomly branched rods, divide as rods and then the rods fragment to spheres as the stationary phase is reached. In a defined liquid medium with glucose as carbon source, the organisms divide entively as spheres at a doubling time of 44 hrs. The addition of l-tyrosine, some fatty acids and tricarboxylic acid cycle intermediates or fructose to the glucose medium caused the cells to grow at considerably faster growth rates (2.8–8.5 hrs doubling times) and to undergo the sphere-rod-sphere growth cycle. Other amino acids, fatty acids or sugars added singly to the glucose medium did not produce the sphere to rod morphology change. Some amino acids when added to the medium in pairs effected sphere to rod morphopoiesis. None of these amino acids alone were effectors. Some of the culture grew as rods and the remainder as spheres when isoleucine and valine were added to the glucose medium. No other amino acid combination tested gave this result. The reason for the mixed growth response was traced to inhomogeneity of the parent culture. The life cycle of N. corallina is illustrated in a series of photomicrographs of two slide cultures.     

Phloem Transport of Amino Acids in Relation to their Cytosolic Levels in Barley Leaves

A comparison of barley (Hordeum vulgare L.) leaves was made between the cytosolic content of amino acids and sucrose as determined by subcellular fractionation and the corresponding concentration in phloem sap, which was collected continuously for up to 6 days from severed aphid stylets. Because amino acids were found to be almost absent from the vacuoles, and because the amino acid patterns in the stroma and cytosol are similar, whole leaf contents could be taken as a measure of cytosolic amino acid levels for a comparison of data during a diurnal cycle. The results show that the pattern of amino acids in the phloem sap was very similar to the pattern in the cytosol. Therefore, we concluded that the overall process of transfer of amino acids from the cytosol of the source cells into the sieve tubes, although carrier mediated, may be a passive process and that the translocation of amino acids via the sieve tubes requires the mass flow of sucrose driven by the active sucrose transport involved by the phloem loading.     

Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-¹⁴C]-palmitate can be taken up from the medium, leading to CO₂ production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.     

Induction of alpha-amylase in barley endosperm by substrate levels of glutamate and aspartate

Incubation of embryoless barley (Hordeum vulgare) half-seeds for 24 hours with 0.1 m glutamate or aspartate resulted in the release of 17 to 48% as much α-amylase as did incubation with 260 mμm gibberellin. With incubation periods of 48 to 51 hours these amino acids were on the average about half as active as response-saturating concentrations of gibberellin, and in some experiments they were essentially as active. Citric acid cycle intermediates, glycolytic pathway intermediates, and cofactors of these pathways failed to induce α-amylase synthesis, while the following compounds were active: asparagine, homoserine, diaminopimelate, isoleucine, methionine, glutamine, ornithine, citrulline, argininosuccinate, and δ-aminolevulinate. However, threonine, lysine, β-alanine, alanine, γ-aminobutyrate, α-ketobutyrate, proline, arginine, glycine, leucine, and putrescine were inactive. Two patterns were noted in the list of active and inactive compounds: (a) all of the active compounds contain an amino group and are biosynthetically derived from citric acid cycle intermediates; and (b) biosynthetic precursors of the amino acids arginine, proline, threonine, and lysine were active whereas these amino acids were not.     

Seasonal and within-plant gradients in the intramolecular carbon isotopic composition of amino acids of Spartina alterniflora

Autotrophy and heterotrophy create different patterns of carbon flux through the central metabolic pathway. One consequence of these different fluxes is that the α-carboxyl carbon of amino acids is derived from different carbon sources and has a different isotopic composition under autotrophic and heterotrophic conditions. In Spartina alterniflora, a C4 grass and a common and ecologically important component of coastal ecosystems, the isotopic composition of bulk acid hydrolyzable carbon and total amino acid carboxyl carbon were compared over a seasonal cycle. The isotopic composition of plants varied significantly between aboveground and below ground tissues, and the δ¹³C of both hydrolyzable organic carbon and total amino acid carboxyl carbon showed significant variation among seasons. The isotopic heterogeneity within amino acids was used to infer seasonal changes in source/sink relationships for amino acid carbon among plant organs. Comparison of the intramolecular isotope data for Spartina and C3 freshwater marsh plants indicates that the patterns and processes inferred for Spartina are not unique to this taxon.     

Analysis of 26 amino acids in human plasma by HPLC using AQC as derivatizing agent and its application in metabolic laboratory

The present study reports the simultaneous analysis of 26 physiological amino acids in plasma along with total cysteine and homocysteine by high-performance liquid chromatography (HPLC) employing 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as precolumn derivatizing reagent. Separations were carried out using Lichrospher 100 RP-18e (5 μm) 250 × 4.0 mm column connected to 100 CN 4.0 × 4.0 mm guard column on a quaternary HPLC system and run time was 53 min. Linearity of the peak areas for different concentrations ranging from 2.5 to 100 pmol/μL of individual amino acids was determined. A good linearity (R ² > 0.998) was achieved in the standard mixture for each amino acid. Recovery of amino acids incorporated at the time of derivatization ranged from 95 to 106 %. Using this method we have established the normative data of amino acids in plasma, the profile being comparable to the range reported in literature and identified cases of classical homocystinuria, cobalamin defect/deficiency, non-ketotic hyperglycinemia, hyperprolinemia, ketotic hyperglycinemia, urea cycle defect and maple syrup urine disease.     

Mechanisms of amino acid-mediated lifespan extension in Caenorhabditis elegans

Background

Little is known about the role of amino acids in cellular signaling pathways, especially as it pertains to pathways that regulate the rate of aging. However, it has been shown that methionine or tryptophan restriction extends lifespan in higher eukaryotes and increased proline or tryptophan levels increase longevity in C. elegans. In addition, leucine strongly activates the TOR signaling pathway, which when inhibited increases lifespan.

Results

Therefore each of the 20 proteogenic amino acids was individually supplemented to C. elegans and the effects on lifespan were determined. All amino acids except phenylalanine and aspartate extended lifespan at least to a small extent at one or more of the 3 concentrations tested with serine and proline showing the largest effects. 11 of the amino acids were less potent at higher doses, while 5 even decreased lifespan. Serine, proline, or histidine-mediated lifespan extension was greatly inhibited in eat-2 worms, a model of dietary restriction, in daf-16/FOXO, sir-2.1, rsks-1 (ribosomal S6 kinase), gcn-2, and aak-2 (AMPK) longevity pathway mutants, and in bec-1 autophagy-defective knockdown worms. 8 of 10 longevity-promoting amino acids tested activated a SKN-1/Nrf2 reporter strain, while serine and histidine were the only amino acids from those to activate a hypoxia-inducible factor (HIF-1) reporter strain. Thermotolerance was increased by proline or tryptophan supplementation, while tryptophan-mediated lifespan extension was independent of DAF-16/FOXO and SKN-1/Nrf2 signaling, but tryptophan and several related pyridine-containing compounds induced the mitochondrial unfolded protein response and an ER stress response. High glucose levels or mutations affecting electron transport chain (ETC) function inhibited amino acid-mediated lifespan extension suggesting that metabolism plays an important role. Providing many other cellular metabolites to C. elegans also increased longevity suggesting that anaplerosis of tricarboxylic acid (TCA) cycle substrates likely plays a role in lifespan extension.

Conclusions

Supplementation of C. elegans with 18 of the 20 individual amino acids extended lifespan, but lifespan often decreased with increasing concentration suggesting hormesis. Lifespan extension appears to be caused by altered mitochondrial TCA cycle metabolism and respiratory substrate utilization resulting in the activation of the DAF-16/FOXO and SKN-1/Nrf2 stress response pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0167-2) contains supplementary material, which is available to authorized users.