14C-labeled Amino Acids Research Articles

Soil nitrogen and phosphorus regulate decomposition of organic nitrogen compounds in the rothamsted experiment

The effects of long-term N and P fertilisation on soil organic nitrogen (SON) turnover are unclear. We sampled soils fertilised by N and/or P since 1843 at Rothamsted Research to investigate the effects of long-term N and P fertilisation on high- or low-molecular-weight SON decomposition and gross N mineralisation. Short-term assays with added 14C-labelled proteins, peptides, and amino acids and gross organic N mineralisation were measured, and the microbial communities and functions were also assessed in parallel. Long-term N fertilisation increased the contents of soil extractable organic N and peptides but decreased organic N-hydrolysing enzyme activities (N-acetyl-β-D-glucosaminidase and leucine aminopeptidase). Fertilisation with P and N accelerated and reduced the decomposition of 14C-labelled organic N compounds, respectively. The decomposition of peptides and amino acids, as labile SON components, was mainly regulated by soil P content because microbial biomass and activity were more sensitive to P fertilisation than to N fertilisation. Gross NH4+/NO3− production and consumption were accelerated by 41%–60% under N fertilisation but remained unchanged under P fertilisation compared to the unfertilised treatment. Metagenomic sequencing showed that N fertilisation increased microbial diversity and enriched functional genes associated with organic N decomposition, compared to the unfertilised treatment. P fertilisation had no effect on the abundance of these functional genes. In agricultural practices, it is essential to comprehensively consider the interaction between N and P fertilisation to optimise the cycling and utilisation of SON.

Cell-free synthesis of proteins with selectively 13C-labelled methyl groups from inexpensive precursors.

The novel eCell system maintains the activity of the entire repertoire of metabolic Escherichia coli enzymes in cell-free protein synthesis. We show that this can be harnessed to produce proteins with selectively C-labelled amino acids from inexpensive C-labelled precursors. The system is demonstrated with selective C labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis-trans isomerase B. Starting from 3-C-pyruvate, C-HSQC cross-peaks are obtained devoid of one-bond C-C scalar couplings. Starting from 2-C-methyl-acetolactate, single methyl groups of valine and leucine are labelled. Labelling efficiencies are 70 % or higher, and the method allows us to produce perdeuterated proteins with protonated methyl groups in a residue-selective manner. The system uses the isotope-labelled precursors sparingly and is readily scalable.

In vitro evaluation of (S)-2-amino-3-[3-(2-18F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid (18F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

Background(S)-2-amino-3-[3-(2-18F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid (18F-FIMP) as a promising PET probe for imaging the tumor-specific L-type amino acid transporter (LAT) 1. Our previous study revealed that 18F-FIMP had a higher affinity for LAT1 than for LAT2 abundantly expressed even in normal cells. 18F-FIMP showed high accumulation in LAT1-positive tumor tissues and low accumulation in inflamed lesions in tumor-bearing mice. However, the affinity of 18F-FIMP for other amino acid transporters was not determined yet. Here, we aimed to determine whether 18F-FIMP has affinity for other tumor-related amino acid transporters, such as sodium- and chloride-dependent neutral and basic amino acid transporter B(0 +) (ATB0,+), alanine serine cysteine transporter 2 (ASCT2), and cystine/glutamate transporter (xCT). ProceduresCells overexpressing LAT1, ATB0,+, ASCT2, or xCT were established by the transfection of expression vectors for LAT1, ATB0,+, ASCT2, or xCT. Protein expression levels were determined by western blot and immunofluorescent analyses. Transport function was evaluated by a cell-based uptake assay using 18F-FIMP and 14C-labeled amino acids as substrates.ResultsIntense signals were observed only for expression vector-transfected cells on western blot and immunofluorescent analyses. These signals were strongly reduced by gene-specific small interfering ribonucleic acid treatment. The uptake values for each 14C-labeled substrate were significantly higher in the transfected cells than in the mock-transfected cells and were significantly inhibited by the corresponding specific inhibitors. The 18F-FIMP uptake values were significantly higher in the LAT1- and ATB0,+-overexpressing cells than in the corresponding mock cells, but no such increase was seen in the ASCT2- or xCT-overexpressing cells. These 18F-FIMP uptake values were significantly decreased by the specific inhibitors for LAT1- and ATB0,+.ConclusionsWe demonstrated that 18F-FIMP has affinity not only for LAT1, but also for ATB0,+. Our results may be helpful for understanding the mechanisms of the whole-body distribution and tumor accumulation of 18F-FIMP.

HPLC-Parallel accelerator and molecular mass spectrometry analysis of 14C-labeled amino acids

Accelerator mass spectrometry (AMS) is the method of choice for quantitation of low amounts of 14C-labeled biomolecules. Despite exquisite sensitivity, an important limitation of AMS is its inability to provide structural information about the analyte. This limitation is not critical when the labeled compounds are well-characterized prior to AMS analysis. However, analyte identity is important in other experiments where, for example, a compound is metabolized and the structures of its metabolites are not known. We previously described a moving wire interface that enables direct AMS measurement of liquid sample in the form of discrete drops or HPLC eluent without the need for individual fraction collection, termed liquid sample-AMS (LS-AMS). We now report the coupling of LS-AMS with a molecular mass spectrometer, providing parallel accelerator and molecular mass spectrometry (PAMMS) detection of analytes separated by liquid chromatography. The repeatability of the method was examined by performing repeated injections of 14C-labeled tryptophan, and relative standard deviations of the 14C peak areas were ≤10.57% after applying a normalization factor based on a standard. Five 14C-labeled amino acids were separated and detected to provide simultaneous quantitative AMS and structural MS data, and AMS results were compared with solid sample-AMS (SS-AMS) data using Bland-Altman plots. To demonstrate the utility of the workflow, yeast cells were grown in a medium with 14C-labeled tryptophan. The cell extracts were analyzed by PAMMS, and 14C was detected in tryptophan and its metabolite kynurenine.

Is soluble protein mineralisation and protease activity in soil regulated by supply or demand?

Protein represents a major input of organic matter to soil and is an important source of carbon (C) and nitrogen (N) for microorganisms. Therefore, determining which soil properties influence protein mineralisation in soil is key to understanding and modelling soil C and N cycling. However, the effect of different soil properties on protein mineralisation, and especially the interactions between soil properties, are poorly understood. We investigated how topsoil and subsoil properties affect protein mineralisation along a grassland altitudinal (catena) sequence that contained a gradient in soil type and primary productivity. We devised a schematic diagram to test the key edaphic factors that may influence protein mineralisation in soil (e.g. pH, microbial biomass, inorganic and organic N availability, enzyme activity and sorption). We then measured the mineralisation rate of 14C-labelled soluble plant-derived protein and amino acids in soil over a two-month period. Correlation analysis was used to determine the associations between rates of protein mineralisation and soil properties. Contrary to expectation, we found that protein mineralisation rate was nearly as fast as for amino acid turnover. We ascribe this rapid protein turnover to the low levels of protein used here, its soluble nature, a high degree of functional redundancy in the microbial community and microbial enzyme adaptation to their ecological niche. Unlike other key soil N processes (e.g. nitrification, denitrification), protease activity was not regulated by a small range of factors, but rather appeared to be affected by a wide range of interacting factors whose importance was dependent on altitude and soil depth [e.g. above-ground net primary productivity (NPP), soil pH, nitrate, cation exchange capacity (CEC), C:N ratio]. Based on our results, we hypothesise that differences in soil N cycling and the generation of ammonium are more related to the rate of protein supply rather than limitations in protease activity and protein turnover per se.

Microbial utilization of low molecular weight organic carbon substrates in cultivated peats in response to warming and soil degradation

Peatlands store vast amounts of carbon (C) within the global terrestrial biosphere. Drainage and cultivation of peat soils lead to rapid soil degradation and C losses, and this may worsen under warming as the soils are no longer protected by anaerobic conditions. To predict the rates of soil C loss and design effective mitigation strategies, it is important to understand what controls organic matter mineralization in these soils. Using the 0–10 cm soil depth of thick and thin (degraded) agricultural peat soils, we investigated the fate of low molecular weight organic substrates (LMWOS) and how the microbial biomass consuming these substrates responded to temperature. We incubated the soils under increasing temperatures (4, 10, 20, and 30 °C) for 72 h. Either 14C-labelled glucose or amino acids were added to the soils and their speed of breakdown, partitioning into anabolic/catabolic processes and microbial C use efficiency (CUE) were determined. The total 14CO2 loss from soil increased significantly with increasing temperature during 72-h incubation, regardless of peat layer thickness. Warming altered the dynamics of LMWOS mineralization by changing C allocation and the turnover rate of different microbial C pools. The half-life of LMWOS decreased more than 50% when temperature increased from 4 to 30 °C for both substrates. CUE was always higher for thin than thick peat soil and both declined by 0.002–0.005 °C-1 with increasing temperature. Thin peat decreased substrate C allocation into the fast cycling C pool compared to the thick peat, but had no overall effect on pool turnover rate. Our work suggests that climate warming will accelerate C mineralization and soil loss in drained peat soils, with larger effects expected in thick peat soil. This study provides an important initial step in characterizing the response of the microbial utilization of labile C to temperature change and soil degradation in cultivated peatlands.

Newly depolymerized large organic N contributes directly to amino acid uptake in young maize plants.

The contribution of large molecular size organic nitrogen (N) to plant N uptake is unclear. Soils with and without maize, at three pH levels, were treated with (carbon-14 and -13 (14 C, 13 C), 15 N) triple-labelled >100kDa organic N. After 48h, soil and maize were sampled for bulk and compound specific isotope analysis to study the turnover in soil and plant 13 C and 15 N uptake. Mineralization of >100kDa organic N increased with higher pH only in soil without maize. The >100kDa organic N disappeared rapidly in soils with and without maize, but surprisingly more >100kDa organic N derived amino acids remained in soil with than without maize - most likely in the microbial biomass. Total 15 N uptake in maize increased with higher soil pH. The organic N uptake was estimated to account for 20-30% of the total 15 N uptake. Organic N uptake was confirmed by the presence of 13 C-labelled amino acids in maize roots. The study suggests that the importance of plant organic N uptake increases when N is derived from complex molecules such as proteins compared to studies using single amino acids as N source, and that rhizosphere microorganisms increase anabolic utilization of organic N compared to microorganisms in the bulk soil.

Another bottleneck for nitrogen mineralization in temperate forest soils: Arginine metabolism in microorganisms

Soil nitrogen (N) mineralization is generally limited by microbial N assimilation when microorganisms are exposed to substrates with high carbon-to-nitrogen (C/N) ratios. We hypothesized that microbial N release is also limited by repression of arginine-degrading activity in forest soils with the high C/N ratios. We analyzed the microbial assimilation and mineralization rates of 14C-labeled amino acid mixture, arginine, ornithine, and urea added to a variety of forest and cropland soils. The proportions of amino acid mixture derived 14C incorporated into microbial biomass (MB14C) in a 24-h incubation (3.7–20.4%) increased with soil C/N ratio and an increase in MB14C retards inorganic N release in the forest soils. Arginine mineralization displays a contrary pattern to amino acid derived MB14C and decreased with increasing soil C/N ratio. The reduced arginine-degrading activity in the forest soils with the high C/N ratios is consistent with general microbial N assimilation associated with growth, but it also correlates with the enhanced fungi-specific N preservation (e.g., recycling of ammonium in urea-ornithine cycle and accumulation of arginine or ornithine in vacuoles). Arginine or ornithine degradation is one of slower amino acid degradation pathways and potentially retards N mineralization in N-limited forest soils.

Peanut RNA Helicase AhRH47 Sustains Protein Synthesis Under Stress and Improves Stress Adaptation in Arabidopsis

Stress responsive RNA helicases are involved in translation initiation sustain protein synthesis. In this study, a stress responsive DEAD box RNA helicase, AhRH47 from peanut cDNA library was identified and characterised during stress. In silico analysis of AhRH47 showed the nine conserved motifs characteristic of an RNA helicase. The phylogenetic and amino acid sequence alignment analyses revealed that AhRH47 is highly homologous to an important DEAD box RNA helicase (eIF4A), which is involved in translation initiation. AhRH47 is stress responsive, being highly expressed under salinity and moisture stress, which is induced to a lesser extent under PEG and ABA treatments. Constitutive overexpression of AhRH47 in Arabidopsis conferred enhanced tolerance to salinity and mannitol-induced stresses. In addition, the transgenic plants showed improved tolerance under moisture stress and exhibited improved recovery growth on stress alleviation. Overexpressing plants showed increased 14C-labelled amino acids incorporation in to protein especially under stress condition. The results suggest AhRH47 transgenic lines maintained higher protein synthesis under stress and thus improved adaptation to osmotic and desiccation stresses.

Synthesis and Preliminary Evaluation of 5-[18F]fluoroleucine.

Amino acid transporters, such as LAT1, are overexpressed in aggressive prostate and breast carcinomas, directly influencing pathways of growth and proliferation. The purpose of this study was to synthesize and characterize a novel 18F labeled leucine analog, 5-[18F]fluoroleucine, as a potential imaging agent for aggressive tumors which may not be amenable to imaging by FDG PET. 5-fluoroleucine was synthesized and characterized, and its 18F-labeled analog was synthesized from a mesylate precursor. First, breast cancer cell line assays were performed to evaluate uptake of 3H- or 14C-labeled L-leucine and other essential amino acids. Both L-leucine and 5- [18F]fluoroleucine were tested for uptake and accumulation over time, and for uptake via LAT1. Biodistribution studies were performed to estimate radiation dosimetry for human studies. Small animal PET / CT studies of a breast cancer were performed to evaluate in vivo 5-[18F]fluoroleucine tumor uptake. Breast cancer cell lines showed increasing high net accumulation of L-[14C]leucine. Both L-leucine and 5-[18F]fluoroleucine showed increasing uptake over time in in vitro tumor cell assays, and uptake was also shown to occur via LAT1. The biodistribution study of 5-[18F]fluoroleucine showed rapid renal excretion, no significant in vivo metabolism, and acceptable dosimetry for use in humans. In vivo small animal PET / CT imaging of a breast cancer xenograft showed uptake of 5- [18F]fluoroleucine in the tumor, which progressively increased over time. 5-[18F]fluoroleucine is a leucine analog which may be useful in identifying tumors with high or upregulated expression of amino acid transporters, providing additional information that may not be provided by FDG PET.

Mineralisation and sorption of dissolved organic nitrogen compounds in litter and soil from sugarcane fields

Dissolved organic nitrogen (DON) represents an important soluble nutrient pool in soil, however, little is known about the dynamics of DON in the litter and topsoil of Brazilian sugarcane (Saccharum spp.) fields, particularly those that are harvested mechanically, without burning. Therefore, the aim of this study was to determine the microbial mineralisation and sorption affinity of DON compounds in litter and soil from the litter-soil transition zone of two sugarcane plantations located in southeastern Brazil. We directly measured the C mineralisation of 14C-labelled amino acids (mix of 17 amino acids), peptides (l-dialanine and l-trialanine), urea, and protein (isolated from tobacco leaves) by capturing 14CO2 evolved from the litter and soil over 168 h. A sorption assay was performed using the same treatments. We found differences in the organic and mineral N pools of the litter and soil, as well as in microbial community composition. Except for protein in the soil, the DON compounds were taken up rapidly by microbes. However, the C use efficiency was higher for the amino acid mix than for the peptides and urea, indicating more rapid post-uptake catabolism (with subsequent mineralisation as 14CO2) of both compounds. In addition, protein had the highest sorption affinity, especially in soil, and the weak sorption affinity of the amino acids, peptides, and urea indicates moderate bioavailability of these fractions to microbes and plants. We conclude that strong sorption of protein to the solid phase limits its bioavailability and represents a rate limiting step in DON turnover.

Modulation of (14) C-labeled glucose metabolism by zinc during aluminium induced neurodegeneration.

Aluminium (Al) is one of the most prominent metals in the environment and is responsible for causing several neurological disorders, including Alzheimer's disease. On the other hand, zinc (Zn) is an essential micronutrient that is involved in regulating brain development and function. The present study investigates the protective potential of Zn in the uptake of (14) C-labeled amino acids and glucose and their turnover in rat brain slices during Al intoxication. Male Sprague Dawley rats (140-160 g) were divided into four different groups: normal control, Al treated (100 mg/kg body weight/day via oral gavage), Zn treated (227 mg/liter in drinking water), and Al + Zn treated. Radiorespirometric assay revealed an increase in glucose turnover after Al exposure that was attenuated after Zn treatment. Furthermore, the uptake of (14) C-labeled glucose was increased after Al treatment but was appreciably decreased upon Zn supplementation. In addition, the uptakes of (14) C-lysine, (14) C-leucine, and (14) C-aspartic acid were also found to be elevated following Al exposure but were decreased after Zn treatment. Al treatment also caused alterations in the neurohistoarchitecture of the brain, which were improved after Zn coadministration. Therefore, the present study suggests that Zn provides protection against Al-induced neurotoxicity by regulating glucose and amino acid uptake in rats, indicating that Zn could be a potential candidate for the treatment of various neurodegenerative disorders.

Soil nitrogen pools and turnover in native woodland and managed pasture soils

Dissolved organic nitrogen (DON) is a significant nitrogen (N) pool in most soils and is considered to be important for N cycling. The present study focused on paired sites of native remnant woodland and managed pasture at three locations in south-eastern Australia. Improved understanding of N cycling is important for assessing the impact of agriculture on soil processes and can guide conservation and restoration soil management strategies to maintain remnant native woodland systems, which currently exist as small pockets of woodland within extensive managed pasture landscapes. Organic and inorganic N pools were quantified, as well as the rates of amino acid and peptide mineralisation in the paired native woodland and managed pasture systems. Soil DON dominated the soil N pool in both land uses, and the proportion of DON to other N pools was greatest at the most N-limited site (up to ∼70% of extractable N). In both land uses soil ammonium and free amino acid concentrations were similar (∼20% of extractable N), and soil nitrate formed the smallest N pool (<∼5% of extractable N). Mineralisation of 14C-labelled amino acid and peptide substrates was rapid (<3 h), and more amino acid was respired than peptide in both the native woodland and managed pasture soils. Soil C:N ratio was important in separating site and land use differences, and contrasting relationships between soil physico-chemical properties and organic N uptake rates were identified across sites and land uses.

Uptake of an amino acid (alanine) and its peptide (trialanine) by the saltmarsh halophytes Salicornia europaea and Aster tripolium and its potential role in ecosystem N cycling and marine aquaculture wastewater treatment

Dissolved organic nitrogen (DON) represents a significant reservoir of potentially plant available nitrogen (N) in saltmarsh ecosystems and wastewater treatment streams. Here we investigated the capacity of two halophytic plants used in wastewater remediation, Salicornia europaea and Aster tripolium, to access soluble organic forms of N when grown in water or soil-based culture. Incubation experiments with 15N13C- and 14C-labelled alanine and trialanine, 15NH4+ and 15NO3− indicated that both S. europaea and A. tripolium can take up and assimilate amino acids (alanine) and oligopeptides (trialanine), in addition to NH4+ and NO3−. In hydroponic solutions the uptake of alanine-N and trialanine-N was comparable to that of NO3−, but lower than NH4+. Comparison of the 13C and 15N ratios in the plant tissues suggested that at least 70% of the alanine and trialanine was taken up intact from solution. Plant capture of exogenously applied N by the soil-grown plants was much lower than in the hydroponically-grown plants. We ascribe this reduced rate of plant N uptake to increased microbial competition for DON, lower rates of N delivery to the root surface (from reduced mass flow and diffusion) and sorption of N solutes to the solid matrix. 14C-labelled amino acid and peptides indicated rapid assimilation of the DON once taken up by the plants. Our results strongly suggest that hydroponically grown S. europaea and A. tripolium are capable of taking up DON at high rates showing that it is highly pertinent to consider DON removal in the phytoremediation of wastewater. In addition, this is the first indication of direct peptide uptake by halophytic plants. We conclude that halophytes have evolved the capacity to take up a wide range of N compounds to satisfy their N demand when growing in N-limiting saltmarsh environments and that this intrinsic potential can be harnessed for wastewater treatment.

Reliable identification of cross-linked products in protein interaction studies by 13C-labeled p-benzoylphenylalanine.

We describe the use of the (13)C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a (13)C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.

Selected amino acids uptake by microorganisms in soil of differently managed mountain meadows

This work was aimed at determination of the effect of 13 years abandonment of previously long-term mown mountain meadows on uptake of L-glutamic acid (14CO2H[14CH2]2[NH2]14CO2H) and L-alanine (14CH314CH[NH2]14CO2H) by microbial community of Ap horizon (3–13 cm). The study plots has been located near to the experimental stand “Bílý Kříž“ which is located in the Moravian-Silesian Beskids Mountains (N 49°30’17”, E 18°32’28”), on a slope with an elevation of 825–860 m a. s. l. and southeast orientation, and soil classified as an Oxyaquic Hapludalf. The study was performed to test method for measurement of 14C-labelled amino acids uptake by soil microbial community and to increase knowledge on particular processes of N-transformation in soil of these ecosystems. The results obtained in this work showed that 13 years abandonment of mountain meadow did not significantly (P &gt; 0.05) influence rate of glutamic acid or alanine uptake by soil microbial community. Further research including determination of amino acids use by soil microbial biomass with expression of their partitioning between production of new microbial biomass and energy metabolism is necessary.

Influence of mineral characteristics on the retention of low molecular weight organic compounds: A batch sorption–desorption and ATR-FTIR study

Batch experiments were conducted to evaluate the sorption–desorption behaviour of 14C-labelled carboxylic acids (citric and oxalic) and amino acids (glutamic, alanine, phenylalanine and lysine) on pure minerals (kaolinite, illite, montmorillonite, ferrihydrite and goethite). The sorption experiments were complemented by ATR-FTIR spectroscopy to gain possible mechanistic insight into the organic acids–mineral interactions. In terms of charge, the organic solutes ranged from strongly negative (i.e., citric) to positively charged solutes (i.e., lysine); similarly the mineral phases also ranged from positively to negatively charged surfaces. In general, sorption of anionic carboxylic and glutamic acids was higher compared to the other compounds (except lysine). Cationic lysine showed a stronger affinity to permanently charged phyllosilicates than Fe oxides. The sorption of alanine and phenylalanine was consistently low for all minerals, with relatively higher sorption and lower desorption of phenylalanine than alanine. Overall, the role of carboxylic functional groups for the sorption and retention of these carboxylic and amino acids on Fe oxides (and kaolinite) and of amino group on 2:1 phyllosilicates was noticeable. Mineral properties (surface chemistry, specific surface area), chemistry of the organic compounds (pKa value, functional groups) and the equilibrium pH of the system together controlled the differences in sorption–desorption patterns. The results of this study aid to understand the effects of mineralogical and chemical factors that affect naturally occurring low molecular weight organic compounds sorption under field conditions.

Lysosomal Amino Acid Efflux Assay

As the cellular “recycling” organelle, lysosomes break down proteins into amino acids, which are then transported into cytosol for reuse by various amino acid transporters. The transport rate of an amino acid is presumably regulated by cellular conditions such as organelle pH, membrane potential and metabolic states. Because of their intracellular localization and the relative inaccessibility, lysosomal amino acid transporters have been studied largely via indirect measurements. Using lysosome purification and 14C-labeled amino acids, this protocol provides a method to measure the efficiency of specific amino acid transporters on lysosomes., 作为细胞&quot;再循环&quot;细胞器，溶酶体将蛋白质分解成氨基酸，然后将其转运到细胞质中以供各种氨基酸转运蛋白重复使用。 氨基酸的转运速率可能通过细胞条件如细胞器pH，膜电位和代谢状态来调节。 由于它们的细胞内定位和相对不可接近性，溶酶体氨基酸转运蛋白主要通过间接测量进行研究。 使用溶酶体纯化和14 C标记的氨基酸，该协议提供了一种测量特定氨基酸转运蛋白在溶酶体上的效率的方法。

Methods for 11C‐ and 18F‐labelling of amino acids and derivatives for positron emission tomography imaging

The different concepts realized for the synthesis of (11) C- and (18) F-labelled amino acids are summarized. Carbon-11 enables principally authentic radiolabelling of natural occurring amino acids by substituting one of the skeleton carbons by the radionuclide. Fluorine-18 is a foreign element for natural amino acids. Because of its advantageous nuclidic properties for positron emission tomography, however, it becomes increasingly important in molecular imaging, also with amino acid analogues. Especially in the last decade, considerable progress has been made with the radiosynthesis of (18) F-labelled amino acids that are now clinically approved, and thus assure their availability. In contrast, the synthetic possibilities with (11) C-labelled amino acids are more limited because of the short half-life of carbon-11 which also hampers their wide spread use.

Organic nitrogen mineralisation in two contrasting agro-ecosystems is unchanged by biochar addition

Biochar additions to soil have been reported to enhance soil fertility whilst simultaneously storing carbon (C). We tested whether either fresh or field-conditioned (aged) biochar amendment to two contrasting agricultural soils would alter the mineralisation of organic N compounds. The mineralisation of 14C-labelled amino acids and peptides were determined over 20 days within each soil. An exponential kinetic decay model was subsequently fitted to the mineralisation data. Overall, statistical analysis revealed significant but small differences between the two biochar treatments and the unamended control treatment. We conclude that biochar has very limited impact on the mineralisation rate of low molecular weight dissolved organic N compounds in these agro-ecosystems.