Amino Acid Load Research Articles

Proteomics and metabolomics analyses reveal the cucurbit sieve tube system as a complex metabolic space.

The plant vascular system, and specifically the phloem, plays a pivotal role in allocation of fixed carbon to developing sink organs. Although the processes involved in loading and unloading of sugars and amino acids are well characterized, little information is available regarding the nature of other metabolites in the sieve tube system (STS) at specific sites along the pathway. Here, we elucidate spatial features of metabolite composition mapped with phloem enzymes along the cucurbit STS. Phloem sap (PS) was collected from the loading (source), unloading (apical sink region) and shoot-root junction regions of cucumber, watermelon and pumpkin. Our PS analyses revealed significant differences in the metabolic and proteomic profiles both along the source-sink pathway and between the STSs of these three cucurbits. In addition, metabolite profiles established for PS and vascular tissue indicated the presence of distinct compositions, consistent with the operation of the STS as a unique symplasmic domain. In this regard, at various locations along the STS we could map metabolites and their related enzymes to specific metabolic pathways. These findings are discussed with regard to the function of the STS as a unique and highly complex metabolic space within the plant vascular system.

Pod removal responsive change in phytohormones and its impact on protein degradation and amino acid transport in source leaves of Brassica napus

To characterize the hormonal regulation of nitrogen remobilization from source to pod filling in Brassica napus, the hormonal level, proteolytic process, and amino acid transport were assessed in mature leaves of pod-removed or control at the early pod-filling stage. Pod (sink) removal decreased salicylic acid (SA), and significantly increased jasmonic acid (JA). The SA/JA ratio decreased with pod removal, accompanied by low degradation of foliar proteins and Rubisco content. A significant decrease in protease activity was observed in pod-removed leaves, confirmed by in-gel staining of protease. Pod removal reduced the expression of four amino acid transporter genes (BnAAP1, BnAAP2, BnAAP4, and BnAAP6) in mature leaves and reduced amino acid loading into phloem. These results indicated that a decrease in SA resulting from pod removal down-regulated nitrogen remobilization accompanied by a decrease in proteolytic activity and amino acid transport in mature leaves at the pod-filling stage.

Connecting Source with Sink: The Role of Arabidopsis AAP8 in Phloem Loading of Amino Acids.

Allocation of large amounts of nitrogen to developing organs occurs in the phloem and is essential for plant growth and seed development. In Arabidopsis (Arabidopsis thaliana) and many other plant species, amino acids represent the dominant nitrogen transport forms in the phloem, and they are mainly synthesized in photosynthetically active source leaves. Following their synthesis, a broad spectrum of the amino nitrogen is actively loaded into the phloem of leaf minor veins and transported within the phloem sap to sinks such as developing leaves, fruits, or seeds. Controlled regulation of the source-to-sink transport of amino acids has long been postulated; however, the molecular mechanism of amino acid phloem loading was still unknown. In this study, Arabidopsis AMINO ACID PERMEASE8 (AAP8) was shown to be expressed in the source leaf phloem and localized to the plasma membrane, suggesting its function in phloem loading. This was further supported by transport studies with aap8 mutants fed with radiolabeled amino acids and by leaf exudate analyses. In addition, biochemical and molecular analyses revealed alterations in leaf nitrogen pools and metabolism dependent on the developmental stage of the mutants. Decreased amino acid phloem loading and partitioning to sinks led to decreased silique and seed numbers, but seed protein levels were unchanged, demonstrating the importance of AAP8 function for sink development rather than seed quality. Overall, these results show that AAP8 plays an important role in source-to-sink partitioning of nitrogen and that its function affects source leaf physiology and seed yield.

Serotonergic modulation of resting state default mode network connectivity in healthy women.

The default mode network (DMN) plays a central role in intrinsic thought processes. Altered DMN connectivity has been linked to diminished cerebral serotonin synthesis. Diminished brain serotonin synthesis is further associated with a lack of impulse control and various psychiatric disorders. Here, we investigated the serotonergic modulation of intrinsic functional connectivity (FC) within the DMN in healthy adult females, controlling for the menstrual cycle phase. Eighteen healthy women in the follicular phase (aged 20-31 years) participated in a double-blind controlled cross-over study of serotonin depletion. Acute tryptophan depletion (ATD) and a balanced amino acid load (BAL), used as the control condition, were applied on two separate days of assessment. Neural resting state data using functional magnetic resonance imaging (fMRI) and individual trait impulsivity scores were obtained. ATD compared with BAL significantly reduced FC with the DMN in the precuneus (associated with self-referential thinking) and enhanced FC with the DMN in the frontal cortex (associated with cognitive reasoning). Connectivity differences with the DMN between BAL and ATD in the precentral gyrus were significantly correlated with the magnitude of serotonin depletion. Right medial frontal gyrus and left superior frontal gyrus connectivity differences with the DMN were inversely correlated with trait impulsivity. These findings partially deviate from previous findings obtained in males and underline the importance of gender-specific studies and controlling for menstrual cycle to further elucidate the mechanism of ATD-induced changes within intrinsic thought processes.

Recapitulation of metabolic defects in a model of propionic acidemia using patient-derived primary hepatocytes.

Propionic acidemia (PA) is a disorder of intermediary metabolism with defects in the alpha or beta subunits of propionyl CoA carboxylase (PCCA and PCCB respectively) enzyme. We previously described a liver culture system that uses liver-derived hemodynamic blood flow and transport parameters to restore and maintain primary human hepatocyte biology and metabolism utilizing physiologically relevant milieu concentrations. In this study, primary hepatocytes isolated from the explanted liver of an 8-year-old PA patient were cultured in the liver system for 10 days and evaluated for retention of differentiated polarized morphology. The expression of PCCA and PCCB was assessed at a gene and protein level relative to healthy donor controls. Ammonia and urea levels were measured in the presence and absence of amino acid supplements to assess the metabolic consequences of branched-chain amino acid metabolism in this disease. Primary hepatocytes from the PA patient maintained a differentiated polarized morphology (peripheral actin staining) over 10 days of culture in the system. We noted lower levels of PCCA and PCCB relative to normal healthy controls at the mRNA and protein level. Supplementation of branched-chain amino acids, isoleucine (5mM) and valine (5mM) in the medium, resulted in increased ammonia and decreased urea in the PA patient hepatocyte system, but no such response was seen in healthy hepatocytes or patient-derived fibroblasts. We demonstrate for the first time the successful culture of PA patient-derived primary hepatocytes in a differentiated state, that stably retain the PCCA and PCCB enzyme defects at a gene and protein level. Phenotypic response of the system to an increased load of branched-chain amino acids, not possible with fibroblasts, underscores the utility of this system in the better understanding of the molecular pathophysiology of PA and examining the effectiveness of potential therapeutic agents in the most relevant tissue.

Sweat facilitated losses of amino acids in Standardbred horses and the application of supplementation strategies to maintain condition during training

Little is known about the amino acid composition of horse sweat, but significant fluid losses can occur during exercise with the potential to facilitate substantial nutrient losses. Sweat and plasma amino acid compositions for Standardbred horses were assessed to determine losses during a standardised training regime. Two cohorts of horses 2013 (n=5) and 2014 (n=6) were assessed to determine baseline levels of plasma and sweat amino acids. An amino acid supplement designed to counter losses in sweat during exercise was provided after morning exercise daily for 5 weeks (2013, n=5; 2014, n=4). After the supplementation period, blood and sweat samples were collected to assess amino acid composition changes. From baseline assessments of sweat in both cohorts, it was found that serine, glutamic acid, histidine and phenylalanine were present at up to 9 times the corresponding plasma concentrations and aspartic acid at 0-2.2 μmol/l in plasma was measured at 154-262 μmol/l in sweat. In contrast, glutamine, asparagine, methionine and cystine were conserved in the plasma by having lower concentrations in the sweat. The predominant plasma amino acids were glycine, glutamine, alanine, valine, serine, lysine and leucine. As the sweat amino acid profile did not simply reflect plasma composition, it was proposed that mechanisms exist to generate high concentrations of certain amino acids in sweat whilst selectively preventing the loss of others. The estimated amino acid load in 16 l of circulating plasma was 3.8-4.3 g and the calculated loss via sweat during high intensity exercise was 1.6-3.0 g. Following supplementation, total plasma amino acid levels from both cohorts increased from initial levels of 2,293 and 2,044 µmol/l to post-supplementation levels of 2,674 and 2,663 µmol/l respectively (P<0.05). It was concluded that the strategy of providing free amino acids immediately after exercise resulted in raising resting plasma amino acid levels.

Arginine behaviour after arginine or citrulline administration in older subjects.

Arginine (ARG) and its precursor citrulline (CIT) are popular dietary supplements, especially for the elderly. However, age-related reductions in lean body mass and alterations in organ functions could change their bioavailability. Pharmacokinetics and tolerance to amino acid (AA) loads are poorly documented in elderly subjects. The objective here was to characterise the plasma kinetics of CIT and ARG in a single-dosing study design. Eight fasting elderly men underwent two separate isomolar oral loading tests (10 g of CIT or 9·94 g of ARG). Blood was withdrawn over an 8-h period to measure plasma AA concentrations. Only CIT, ornithine and ARG plasma concentrations were changed. Volume of distribution was not dependent on AA administered. Conversely, parameters related to ARG kinetics were strongly dependent on AA administered: after ARG load, elimination was higher (ARG>CIT; P=0·041) and admission period+time at peak concentration was lower (ARG<CIT; P=0·033), and the combination of both phenomena results in a marked increase in ARG availability when CIT was administered (ARG<CIT; P=0·033) compared with ARG administration itself. In conclusion, a single CIT administration in the elderly is safe and well tolerated, and CIT proves to be a better in vivo ARG precursor than ARG itself in healthy elderly subjects.

Increasing lean muscle mass: nutritional and periodization strategies

Increases in skeletal muscle mass arises as a result of positive net protein balance, such that muscle protein synthesis (MPS) exceeds muscle protein breakdown (MPB). Increased mechanical loading and provision of high quality amino acids are potent and independent stimulators of MPS through activation of key cell signaling pathways involving the mTOR-p70S6K signaling axis. Importantly, the combination of resistance exercise and dietary protein intake elevate MPS over and above their independent effects, thus highlighting that a well formulated resistance training programme (incorporating multiple sets to failure) and increased dietary protein intake (spread evenly throughout the day) should form the basis of training and nutritional strategies. However, given that many elite athletes (such as rowers) may simultaneously be training to develop endurance as well as strength, it is also important to consider the periodization of endurance type training sessions alongside high volume resistance sessions. In this regard the signaling pathway known to regulate the endurance phenotype (i.e. the AMPK-PGC-1 axis) may potentially attenuate the activation of growth related pathways thereby mediating a training interference effect by which lean mass growth is negated. As such, careful consideration should also be given to the training structure and nutrient availability both within and between days so as to maximize training adaptation and recovery. In this presentation, the author presents data from both his own laboratory and others that briefly outlines the molecular regulation of muscle mass growth and endurance adaptations before providing nutritional and training periodization strategies such that both aspects of training adaptation may be developed simultaneously.

Macroporous chitosan hydrogels: Effects of sulfur on the loading and release behaviour of amino acid-based compounds

Chitosan is a biodegradable, biocompatible polymer of natural origin widely applied to the preparation of functional hydrogels suitable for controlled release of drugs, peptides and proteins. Non-covalent interactions, expecially ionic interactions, are the main driver of the loading and release behaviour of amino acids or peptides from chitosan hydrogels. With the aim to improve the understanding of the mechanisms governing the behaviour of chitosan hydrogels on peptide uptake and delivery, in this paper the attention was focused on the role played by sulfur on the interactions of chitosan hydrogels with sulfur-containing amino acids (AA) and peptides. Hence, loading and release experiments on cysteine, cystine and glutathione (SH containing amino acid, dipeptide and tripeptide, respectively) as well as on glycine and valine as apolar amino acids were carried out. For these puroses, chitosan hydrogels were prepared in an easy and reproducible manner by a freeze-gelation process on a poly-l-lysine coated support. The hydrogel surface pore size, uniformity and distribution were tested. Optimal results (D50=26±4μm) were obtained by using the poly-l-lysine positively-charged surface. The loading results gathered evidenced that the sulfur-containing molecules presented an increased absorption both in terms of rate and extent by chitosan hydrogels with respect to nonpolar amino acids, mainly due to ionic and hydrogen bond interactions. ATR-FTIR analysis carried out on chitosan hydrogels, with and without the AA related compounds to study putative interactions, supported these apparent sulfur-dependent results. Finally, chitosan hydrogels displayed excellent retention capabilities (AA release <5%) for all AA, strongly supporting the use of chitosan hydrogels as matrix for controlled drug release.

Effects of serotonin depletion on punishment processing in the orbitofrontal and anterior cingulate cortices of healthy women

Diminished synthesis of the neurotransmitter serotonin (5-HT) has been linked to disrupted impulse control in aversive contexts. However, the neural correlates underlying a serotonergic modulation of female impulsivity remain unclear. The present study investigated punishment-induced inhibition in healthy young women. Eighteen healthy female subjects (aged 20–31) participated in a double-blinded, counterbalanced, placebo-controlled, within subjects, repeated measures study. They were assessed on two randomly assigned occasions that were controlled for menstrual cycle phase. In a randomized order, one day, acute tryptophan depletion (ATD) was used to reduce 5-HT synthesis in the brain. On the other day, participants received a tryptophan-balanced amino acid load (BAL) as a control condition. Three hours after administration of ATD/BAL, neural activity was recorded during a modified Go/No-Go task implementing reward or punishment processes using functional magnetic resonance imaging (fMRI). Neural activation during No-Go trials in punishment conditions after BAL versus ATD administration correlated positively with the magnitude of central 5-HT depletion in the ventral and subgenual anterior cingulate cortices (ACC). Furthermore, neural activation in the medial orbitofrontal cortex (mOFC) and the dorsal ACC correlated positively with trait impulsivity. The results indicate reduced neural sensitivity to punishment after short-term depletion of 5-HT in brain areas related to emotion regulation (subgenual ACC) increasing with depletion magnitude and in brain areas related to appraisal and expression of emotions (mOFC and dorsal ACC), increasing with trait impulsivity. This suggests a serotonergic modulation of neural circuits related to emotion regulation, impulsive behavior, and punishment processing in females.

Standardization of formulations for the acute amino acid depletion and loading tests.

The acute tryptophan depletion and loading and the acute tyrosine plus phenylalanine depletion tests are powerful tools for studying the roles of cerebral monoamines in behaviour and symptoms related to various disorders. The tests use either amino acid mixtures or proteins. Current amino acid mixtures lack specificity in humans, but not in rodents, because of the faster disposal of branched-chain amino acids (BCAAs) by the latter. The high content of BCAA (30-60%) is responsible for the poor specificity in humans and we recommend, in a 50g dose, a control formulation with a lowered BCAA content (18%) as a common control for the above tests. With protein-based formulations, α-lactalbumin is specific for acute tryptophan loading, whereas gelatine is only partially effective for acute tryptophan depletion. We recommend the use of the whey protein fraction glycomacropeptide as an alternative protein. Its BCAA content is ideal for specificity and the absence of tryptophan, tyrosine and phenylalanine render it suitable as a template for seven formulations (separate and combined depletion or loading and a truly balanced control). We invite the research community to participate in standardization of the depletion and loading methodologies by using our recommended amino acid formulation and developing those based on glycomacropeptide.

Amino Acid Modified Macroreticular Anion Exchange Resins for CO&lt;sub&gt;2&lt;/sub&gt; Adsorption

Amino acids were used to modify macroreticular ion exchange resin for the adsorptive capture of CO2. Adsorption isotherms, adsorption rate curves, heats of adsorption, and gas separation performance were determined. It is found that the adsorption capacity of the resin increases significantly after the loading of amino acids. The adsorption progresses rapidly and reaches equilibrium within 3 min. The adsorption capacity decreases with the increasing temperature, and room temperature is a suitable condition that enables high adsorption of CO2. The adsorbent can be promoted by water vapor in removing CO2 effectively from gas mixtures. Ten continuous adsorption–desorption cycles was performed, showing that the adsorbent is of good regeneration performance and the adsorption capacity keeps at about 0.9 mmol/g after regeneration.

Extraction of Apoplastic Wash Fluids and Leaf Petiole Exudates from Leaves of Arabidopsis thaliana

The long-distance translocation of metabolites and mineral elements is crucial for plant growth and reproduction. In most cases, source-to-sink translocation of metabolites and minerals requires their passage through the apoplast, irrespective whether they are transported via the xylem or the phloem. This apoplast-mediated pathway is of particular importance during plant senescence, when photoassimilates as well as organic, inorganic or chelated forms of nutrients are translocated from leaves to fruits or seeds. Recent genetic and physiological studies revealed the involvement of numerous membrane transporters mediating phloem loading of amino acids, sugars, urea or mineral elements. To evaluate the contribution of individual transporters to xylem unloading or phloem loading, the collection of apoplastic fluids and of phloem sap is essential. Here, we describe a method for the extraction of apoplastic fluids and the collection of leaf petiole exudates from Arabidopsis leaves, the latter representing an approximation to the real composition of the phloem sap.

Studying the effects of dietary body weight-adjusted acute tryptophan depletion on punishment-related behavioral inhibition

BackgroundAlterations in serotonergic (5-HT) neurotransmission are thought to play a decisive role in affective disorders and impulse control.ObjectiveThis study aims to reproduce and extend previous findings on the effects of acute tryptophan depletion (ATD) and subsequently diminished central 5-HT synthesis in a reinforced categorization task using a refined body weight–adjusted depletion protocol.DesignTwenty-four young healthy adults (12 females, mean age [SD]=25.3 [2.1] years) were subjected to a double-blind within-subject crossover design. Each subject was administered both an ATD challenge and a balanced amino acid load (BAL) in two separate sessions in randomized order. Punishment-related behavioral inhibition was assessed using a forced choice go/no-go task that incorporated a variable payoff schedule.ResultsAdministration of ATD resulted in significant reductions in TRP measured in peripheral blood samples, indicating reductions of TRP influx across the blood–brain barrier and related brain 5-HT synthesis. Overall accuracy and response time performance were improved after ATD administration. The ability to adjust behavioral responses to aversive outcome magnitudes and behavioral adjustments following error contingent punishment remained intact after decreased brain 5-HT synthesis. A previously observed dissociation effect of ATD on punishment-induced inhibition was not observed.ConclusionsOur results suggest that neurodietary challenges with ATD Moja–De have no detrimental effects on task performance and punishment-related inhibition in healthy adults.

CrossTalk proposal: The dominant mechanism causing disuse muscle atrophy is decreased protein synthesis.

CrossTalk proposal: The dominant mechanism causing disuse muscle atrophy is decreased protein synthesis.

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids.

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic-active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane-localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element-companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.

Accumulation, selection and covariation of amino acids in sieve tube sap of tansy (Tanacetum vulgare) and castor bean (Ricinus communis): evidence for the function of a basic amino acid transporter and the absence of a γ-amino butyric acid transporter

Sieve tube sap was obtained from Tanacetum by aphid stylectomy and from Ricinus after apical bud decapitation. The amino acids in sieve tube sap were analyzed and compared with those from leaves. Arginine and lysine accumulated in the sieve tube sap of Tanacetum more than 10-fold compared to the leaf extracts and they were, together with asparagine and serine, preferably selected into the sieve tube sap, whereas glycine, methionine/tryptophan and γ-amino butyric acid were partially or completely excluded. The two basic amino acids also showed a close covariation in sieve tube sap. The acidic amino acids also grouped together, but antagonistic to the other amino acids. The accumulation ratios between sieve tube sap and leaf extracts were smaller in Ricinus than in Tanacetum. Arginine, histidine, lysine and glutamine were enriched and preferentially loaded into the phloem, together with isoleucine and valine. In contrast, glycine and methionine/tryptophan were partially and γ-amino butyric acid almost completely excluded from sieve tube sap. The covariation analysis grouped arginine together with several neutral amino acids. The acidic amino acids were loaded under competition with neutral amino acids. It is concluded from comparison with the substrate specificities of already characterized plant amino acid transporters, that an AtCAT1-like transporter functions in phloem loading of basic amino acids, whereas a transporter like AtGAT1 is absent in phloem. Although Tanacetum and Ricinus have different minor vein architecture, their phloem loading specificities for amino acids are relatively similar.

Amino acid‐dependent regulation of food intake: is protein more than the sum of its parts?

Amino acid‐dependent regulation of food intake: is protein more than the sum of its parts?

Influence of acute tryptophan depletion on verbal declarative episodic memory in young adult females

Diminished synthesis of the neurotransmitter serotonin (5-HT) in the brain has been linked to disturbed memory processes. The present study investigated the effects of diminished central nervous 5-HT synthesis as achieved by an acute dietary tryptophan depletion (ATD) on verbal declarative episodic memory in young women while controlling for the effects of female sex hormones. Eighteen healthy females (aged 20-31 years) participated in a within-subject repeated measures study, with two separate days of assessment spaced at least one individual menstrual cycle apart. On one day, participants were subjected to ATD, thus lowering central nervous 5-HT synthesis. The other day participants received a tryptophan-balanced amino acid load (BAL = control condition). The study was randomized, counterbalanced and double blind in terms of ATD/BAL administration. Measurements took place in the early follicular phase of the participants' menstrual cycle. Estrogen, FSH and LH levels were assessed at baseline. Verbal declarative episodic memory was assessed using a structured word-learning task. Short-term memory, as indexed by immediate recall, was reduced after ATD intake, whereas delayed recall and recognition after a 25-min delay did not show any differences after intake of ATD or BAL. In young women, verbal short-term memory function was more vulnerable to ATD than consolidation processes. In light of the possible interplay between female sex hormones and 5-HT, further studies comparing different menstrual cycle phases are needed.

The impact of 5-HT on neuropsychological trajectories of adult ADHD

The impact of the neurotransmitter serotonin (5-HT) on symptoms in adult ADHD is underinvestigated. The aim of this study was to test the effects of diminished brain 5-HT synthesis as achieved by acute tryptophan depletion (ATD) on attention, affective prosody and impulsive aggression in male adult patients with ADHD. In a double-blind within subjects' crossover design patients with ADHD (n = 20) and healthy controls (N = 20) completed ATD or a balanced amino acid load (BAL) on either one of two examination days. Neuropsychological testing included the subtest logical memory of the WMS as well as the Point Subtraction Aggression Game. In addition a modified Continuous Performance Test (AX-CPT) and the Tubingen Affect Battery were administered. Lowered rates of reactive aggression were found in the ADHD group under ATD after low provocation. In addition statistical analysis revealed significant shorter reaction times, more errors and omissions in ADHD as well as an interaction ([omissions] x [ATD/BAL] x [Group]).No ATD effect on affective prosody and memory was observed. The present results indicate an involvement of 5-HT in several neuropsychological domains of adult ADHD. Further studies are needed to explore underlying general serotonergic dysfunction in adult ADHD.