Kinetics of amino acid uptake by ectomycorrhizal roots

It is well established that ectomycorrhizal fungi can use amino acids as nitrogen and carbon sources, but data on the kinetic properties of amino acid uptake systems of ectomycorrhizal systems are scarce. Using ¹⁴C-labelled compounds we have determined the kinetics of uptake of amino acids by excised ectomycorrhizal roots for a range of distinct mycorrhizal types from three tree species, beech, spruce, and pine. All mycorrhizal types examined took up amino acids via high-affinity transport systems ( K _M values ranging from 19 to 233 mmol m^–3). A comparative analysis of kinetic parameters for uptake of amino acids and the ammonium analogue methylammonium showed that ectomycorrhizal roots have similar or even higher affinities (lower K _M values) for the amino acids, indicating that absorption of these organic forms of nitrogen (N) can contribute significantly to total N uptake by ectomycorrhizal plants. Analysis of amino acid uptake by ectomycorrhizal roots collected along a European north/south gradient of increasing mineral N pollution from northern Sweden to south Germany revealed no obvious trend in the uptake capabilities for amino acids by ectomycorrhizal roots in relation to the location of the sampling site on this gradient. Rather, the fungal species forming a particular morphotype was the factor determining uptake kinetics. It can therefore be deduced that the species composition of the fungal community will contribute significantly to the functional diversity of a population of mycorrhizal roots.     

Gene expression level shapes the amino acid usages in Prochlorococcus marinus MED4

Prochlorococcus species are the first example of free-living bacteria with reduced genome. Codon and amino acid usages bias of Prochlorococcus marinus MED4 was investigated using all protein coding genes having length greater than or equal to 100 amino acids. Correspondence analysis on relative synonymous codon usage (RSCU) values shows that there is no such influence of translational selection in shaping the codon usage variation among the genes in this organism. However, amino acid usages were markedly different between the highly and lowly expressed genes in this organism and in particular, GC rich amino acids were found to occur significantly higher in highly expressed genes than the lowly expressed genes. Comparative analysis of the homologous genes of Synechococcus sp. WH8102 and Prochlorococcus marinus MED4 shows that amino acids conservation in highly expressed genes is significantly higher than lowly expressed genes. Based on our results we concluded that conservation of GC rich amino acids in the highly expressed genes to its ancestor is the major source of variation in amino acid usages in the organism.     

Diel rhythmicity of lipid-body formation in a coral-Symbiodinium endosymbiosis

The biogenesis of intracellular lipid bodies (LBs) is dependent upon the symbiotic status between host corals and their intracellular dinoflagellates (genus Symbiodinium), though aside from this observation, little is known about LB behavior and function in this globally important endosymbiosis. The present research aimed to understand how LB formation and density are regulated in the gastrodermal tissue layer of the reef-building coral Euphyllia glabrescens. After tissue fixation and labeling with osmium tetroxide, LB distribution and density were quantified by imaging analysis of serial cryo-sections, and a diel rhythmicity was observed; the onset of solar irradiation at sunrise initiated an increase in LB density and size, which peaked at sunset. Both LB density and size then decreased to basal levels at night. On a seasonal timescale, LB density was found to be significantly positively correlated with seasonal irradiation, with highest densities found in the summer and lowest in the fall. In terms of LB lipid composition, only the concentration of wax esters, and not triglycerides or sterols, exhibited diel variability. This suggests that the metabolism and accumulation of lipids in LBs is at least partially light dependent. Ultrastructural examinations revealed that the LB wax ester concentration correlated with the number of electron-transparent inclusion bodies. Finally, there was a directional redistribution of the LB population across the gastroderm over the diel cycle. Collectively, these data reveal that coral gastrodermal LBs vary in composition and intracellular location over diel cycles, features which may shed light on their function within this coral–dinoflagellate mutualism.     

Modulation of amino acid uptake by TGF-beta in lung myofibroblasts

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway. We examine the effect and interaction of TGF-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho-Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho-Akt levels. LY294002, a PI3-kinase inhibitor, blocked the TGF-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-induced phospho-Akt levels. Inhibition of PI3-kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3-kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.     

Characteristics of amino acid uptake in barley

Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten kinetics. The affinity constant (K _m) was in the range 19.6–33.2 μM. These K _m values are comparable to reported values for soil micro-organisms.     

Diel variation in branchial calcium uptake by the rainbow trout

Branchial Ca uptake varied dielly with a nadir at 1000 hours and two peaks at 1600 and 0400 hours in rainbow trout. This variation profile essentially reflected Ca uptake through the arterio-arterial circulation.     

Intact amino acid uptake by northern hardwood and conifer trees

Empirical and modeling studies of the N cycle in temperate forests of eastern North America have focused on the mechanisms regulating the production of inorganic N, and assumed that only inorganic forms of N are available for plant growth. Recent isotope studies in field conditions suggest that amino acid capture is a widespread ecological phenomenon, although northern temperate forests have yet to be studied. We quantified fine root biomass and applied tracer-level quantities of U–¹³C₂–¹⁵N-glycine, ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ in two stands, one dominated by sugar maple and white ash, the other dominated by red oak, beech, and hemlock, to assess the importance of amino acids to the N nutrition of northeastern US forests. Significant enrichment of ¹³C in fine roots 2 and 5 h following tracer application indicated intact glycine uptake in both stands. Glycine accounted for up to 77% of total N uptake in the oak–beech–hemlock stand, a stand that produces recalcitrant litter, cycles N slowly and has a thick, amino acid-rich organic horizon. By contrast, glycine accounted for only 20% of total N uptake in the sugar maple and white ash stand, a stand characterized by labile litter and rapid rates of amino acid production and turnover resulting in high rates of mineralization and nitrification. This study shows that amino acid uptake is an important process occurring in two widespread, northeastern US temperate forest types with widely differing rates of N cycling.     

Cystine and dibasic amino acid uptake by opossum kidney cells

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.     

Autoradiographic analysis of amino acid uptake by the gill ofMytilus

Summary Autoradiography was used to examine the influence of lateral ciliary activity on the pattern of leucine uptake into isolated gill tissue from the mussel,Mytilus californianus. Metachronal activity of the lateral cilia, normally absent in the in vitro gill, was reestablished by application of 10 μM 5-hydroxytryptamine (5-HT). This treatment produced a 5–7 fold stimulation in the rate of leucine uptake into isolated gills. The treatment with 5-HT did not, however, affect the fractional incorporation of leucine into alcohol insoluble vs alcohol soluble material. Autoradiograms of gills treated with 5-HT showed extensive labelling of frontal, lateral, and abfrontal surfaces of gill filaments compared to the control condition in which label was largely confined to the frontal region of the gill. Quantitative analyses of the autoradiograms revealed a 4-fold increase in the number of silver grains over lateral and abfrontal surfaces compared to control gills. Autoradiograms of gills from intact mussels exposed to³H-leucine showed a pattern of silver grain deposition similar to that observed in in vitro gills treated with 5-HT. It is concluded that the capacity for amino acid transport exists in cells from the frontal, lateral, and abfrontal surfaces of gill filaments, butaccess to dissolved substrates by transport sites on lateral and abfrontal surfaces is dependent upon lateral ciliary activity.     

Effect of ammonia on amino acid uptake by brain microvessels

NH+4 ions, at a concentration (0.25 mM) similar to that found in the plasma of patients with hepatic encephalopathy, cause, in vitro, a significant stimulation of the uptake by brain microvessels of large neutral amino acids, without any effect on the uptake of alpha-methylaminoisobutyric acid, glutamic acid, or lysine. Such a stimulation occurs essentially through an increase of the maximal transport capacity (Vmax) of the saturable component. It is apparently mediated by the intracellular formation of glutamine, which is then exchanged, through the L-system of transport, for large neutral amino acids such as leucine, phenylalanine, or tyrosine. At higher concentrations (greater than or equal to 0.5 mM), NH+4 ions cause also a decrease of carrier affinity for neutral amino acids, which counteracts the stimulatory effect on their uptake.     

Diel periodicity in uptake of nitrate and nitrite by reservoir phytoplankton

Diel periodicity in the uptake of nitrate, and nitrite as measured by the ¹⁵N technique, occurs in reservoir phytoplankton. The time course of changes in the rate of nitrate uptake generally paralleled changes in irradiance. Uptake of nitrate and nitrite occurred in the dark, but at low rates. Periodicity in nitrate uptake needs to be considered in models of primary production where nitrogen is the limiting nutrient.     

Energization of amino acid uptake by system A in cultured human fibroblasts

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.     

Regulation of amino acid uptake in conifers by exogenous and endogenous nitrogen

Although an accumulating amount of research clearly indicates that plants are capable of taking up exogenous amino acids, the actual importance of such organic N sources for plant N nutrition is under debate. In this study, we show that amino acid uptake by Scots pine (Pinus sylvestris L.) is significantly decreased by elevated internal NH(4)(+) levels, while it increases following exposure to exogenous amino acids. Furthermore, amino acid uptake is larger in N-deficient plants than in plants grown with a large access of N. The regulatory pattern of amino acid uptake shows important similarities to the regulation of NO(3)(-) and NH(4)(+) transport as well as to the regulation of yeast amino acid transporters. In addition, our data suggest that uptake may be regulated by factors not originating from N metabolism. The up-regulation of uptake in response to N deficiency suggests that amino acid uptake may be a significant contributor to the N economy of P. sylvestris.     

Transcellular mechanisms of amino acid uptake by distal rat ileum in situ

A 10 cm distal ileal intestinal perfusion technique was employed in Sprague-Dawley rats in situ. The perfused segment was removed, weighed, its surface area measured, homogenized, digested in HNO3 and assayed for L(1-14C)alanine and L-phenyl (1-14C)alanine. Steady state for L-alanine and L-phenylalanine absorption by the intact intestinal segment was observed at 10 and 15 min respectively. Exposure of the intestinal mucosa to 1 mM ouabain showed no effect on amino acid absorption. Preloading the intestinal epithelium with ouabain resulted in approximately 66% and 48% reduction in L-alanine and L-phenylalanine absorption respectively. Removal of Na from the buffer with and without exposure of the mucosa to 1 mM ouabain decreased absorption of L-alanine and L-phenylalanine by approximately 77% and 52% respectively. Removal of Na from the buffer and preloading the intestinal epithelium with ouabain resulted in approximately 85% and 81% reduction in L-alanine and L-phenylalanine absorption respectively. A 5, 10 and 25 fold increase in luminal L-alanine and L-phenylalanine concentration in Na-free choline Krebs Ringer after preloading with ouabain resulted in increase of amino acid absorption of approximately the same order of magnitude. Both an amino acid-carrier mediated transport process and a ouabain resistant Na-dependent-amino acid pump exist at the mucosal side. Both an ouabain sensitive Na-dependent-amino acid pump and an ouabain resistant Na-independent amino acid pump exist at the serosal side. Approximately 15-20% of absorbed amino acids are passively translocated.