Alanine Uptake Research Articles

CD169+ Macrophages Mediate the Immune Response of Allergic Rhinitis Through the Keap1/Nrf2/HO-1 Axis.

CD169+ macrophages are a newly defined macrophage subpopulation that can recognize and bind with other cells through related ligands, playing an essential role in antigen presentation and immune tolerance. However, its role in Allergic Rhinitis (AR) is still unclear. To investigate the characteristics of CD169+ macrophages in AR, this work first detects their expression patterns in the nasal mucosa of clinical patients. These results show a significant increase in CD169+ macrophages in the nasal mucosa of patients with AR. Subsequently, this work establishes an animal AR model using CD169 transgenic mice and compared the advantages of the two models. Moreover, this work also demonstrates the effects of CD169 knockout on eosinophils, Th cells, Treg cells, and the migration of dendritic cells (DCs). In addition, this metabolomic data shows that CD169+ macrophages can upregulate alanine production and increase reactive oxygen species (ROS) levels. This process may be mediated through the Keap1/Nrf2/HO-1 signaling pathway. In addition, this work also finds that SLC38A2 plays an essential role in the process of CD169+ macrophages promoting alanine uptake by DCs. This study confirms that CD169+ macrophages can upregulate their internal alanine production and increase ROS levels through the Keap1/Nrf2/HO-1 axis, playing an irreplaceable role in AR.

Chromium and Methionine Effects on Amino Acid Uptake, Gene Expression, and Protein Abundance in Bovine Satellite Cells

The objective of the study was to evaluate the effects of chromium (Cr) and methionine on myogenic gene expression, GLUT4 and insulin receptor (IR) synthesis, and amino acid uptake in differentiated bovine satellite cells. Cells were treated with 0, 1, or 10 μM of chromium acetate (CrAc) and 0, 1, or 10 mg/mL of methionine, with cells utilized for the amino acid assay receiving 0.1 μM instead of 1 μM of CrAc. The addition of 10 μM of Cr tended to downregulate AMPKα mRNA gene expression (P < 0.1). Myogenin was impacted by methionine and CrAc with high doses and low doses upregulating expression (P < 0.01). The ratio and protein levels of AMPKα to pAMPKα, GLUT4, and IR abundance were not affected (P > 0.1). Increase in time decreased concentrations of leucine, isoleucine, threonine, methionine, phenylalanine, glutamine, ornithine, lysine, and cysteine (P < 0.01) in conditioned media. Over time, alanine increased (P < 0.01). Methionine inclusion increased alanine, proline, glutamic acid, ornithine, and tryptophan concentrations, as well as decreased leucine, isoleucine, and cysteine (P < 0.01). CrAc only affected glutamine and tryptophan, which both increased with CrAc dose, whereas alanine decreased (P < 0.01). Methionine decreased over time within the 10 mg/mL group (P < 0.01). Tryptophan was generally unaffected but decreased in the 0 mg/mL group at 48 h. Cysteine decreased with a decreased dosage of CrAc at 24 h (P < 0.01). Lysine was generally unaffected by this interaction (P > 0.1), with the exception of the 0.1 μM treatment group at 24 h being higher (P < 0.01). CrAc affected the uptake of alanine, glutamine, and tryptophan (P < 0.01). Methionine inclusion increased alanine, proline, glutamic acid, ornithine, and tryptophan, as well as decreased leucine, isoleucine, and cystine (P < 0.01). Increased time saw a significant decrease in leucine, isoleucine, threonine, methionine, phenylalanine, glutamine, ornithine, lysine, and cystine (P < 0.01) and an increase in alanine (P < 0.01).

The NERP-4–SNAT2 axis regulates pancreatic β-cell maintenance and function

Insulin secretion from pancreatic β cells is regulated by multiple stimuli, including nutrients, hormones, neuronal inputs, and local signalling. Amino acids modulate insulin secretion via amino acid transporters expressed on β cells. The granin protein VGF has dual roles in β cells: regulating secretory granule formation and functioning as a multiple peptide precursor. A VGF-derived peptide, neuroendocrine regulatory peptide-4 (NERP-4), increases Ca2+ influx in the pancreata of transgenic mice expressing apoaequorin, a Ca2+-induced bioluminescent protein complex. NERP-4 enhances glucose-stimulated insulin secretion from isolated human and mouse islets and β-cell–derived MIN6-K8 cells. NERP-4 administration reverses the impairment of β-cell maintenance and function in db/db mice by enhancing mitochondrial function and reducing metabolic stress. NERP-4 acts on sodium-coupled neutral amino acid transporter 2 (SNAT2), thereby increasing glutamine, alanine, and proline uptake into β cells and stimulating insulin secretion. SNAT2 deletion and inhibition abolish the protective effects of NERP-4 on β-cell maintenance. These findings demonstrate a novel autocrine mechanism of β-cell maintenance and function that is mediated by the peptide–amino acid transporter axis.

Candidatus Accumulibacter use fermentation products for enhanced biological phosphorus removal

Previous research suggested that two major groups of polyphosphate-accumulating organisms (PAOs), i.e., Ca. Accumulibacter and Tetrasphaera, play cooperative roles in enhanced biological phosphorus removal (EBPR). The fermentation of complex organic compounds by Tetrasphaera provides carbon sources for Ca. Accumulibacter. However, the viability of the fermentation products (e.g., lactate, succinate, alanine) as carbon sources for Ca. Accumulibacter and their potential effects on the metabolism of Ca. Accumulibacter were largely unknown. This work for the first time investigated the capability and metabolic details of Ca. Accumulibacter cognatus clade IIC strain SCUT-2 (enriched in a lab-scale reactor with a relative abundance of 42.8%) in using these fermentation products for EBPR. The enrichment culture was able to assimilate lactate and succinate with the anaerobic P release to carbon uptake ratios of 0.28 and 0.36 P mol/C mol, respectively. In the co-presence of acetate, the uptake of lactate was strongly inhibited, since two substrates shared the same transporter as suggested by the carbon uptake bioenergetic analysis. When acetate and succinate were fed at the same time, Ca. Accumulibacter assimilated two carbon sources simultaneously. Proton motive force (PMF) was the key driving force (up to 90%) for the uptake of lactate and succinate by Ca. Accumulibacter. Apart from the efflux of proton in symport with phosphate via the inorganic phosphate transport system, translocation of proton via the activity of fumarate reductase contributed to the generation of PMF, which agreed with the fact that PHV was a major component of PHA when lactate and succinate were used as carbon sources, involving the succinate-propionate pathway. Metabolic models for the usage of lactate and succinate by Ca. Accumulibacter for EBPR were built based on the combined physiological, biochemical, metagenomic, and metatranscriptomic analyses. Alanine was shown as an invalid carbon source for Ca. Accumulibacter. Instead, it significantly and adversely affected Ca. Accumulibacter-mediated EBPR. Phosphate release was observed without alanine uptake. Significant inhibitions on the aerobic phosphate uptake was also evident. Overall, this study suggested that there might not be a simply synergic relationship between Ca. Accumulibacter and Tetrasphaera. Their interactions would largely be determined by the kind of fermentation products released by the latter.

How do tree species with different successional stages affect soil organic nitrogen transformations?

Organic nitrogen (N) is the most important N component of soil organic matter. However, knowledge on how tree species with different successional stages affect organic N transformations in soils remains limited. To address this issue, we sampled mineral soils (0–10 cm) under monocultures composed of tree species from different successional stages, including early (black alder and silver birch), early to mid (sycamore and European ash), and late (sweet chestnut, pedunculate oak and European beech), and measured the potential protease activity, the microbial uptake and respiration of 14C-labeled organic N (l-alanine and l-trialanine), and the mineralization of l-alanine N. The activities of alanine aminopeptidase and leucine aminopeptidase (153.8–341.9 and 91.6–147.9 nmol/g/h, respectively), the half-life of the uptake of alanine and trialanine (26.7–39.6 and 60.8–78.6 min, respectively), the half-life of the mineralization of alanine and trialanine (1.98–2.45 and 2.98–4.13 h, respectively) by soil microbes were altered by tree species from different successional stages, systematically changing the transformation chain of soil organic N. The turnover rates of soil organic N under trees of early to late successional stage appeared to decrease and the half-life appeared to increase significantly. The C:N ratio of soil microbial biomass was positively related to the half-life of 14C-labeled alanine and trialanine mineralization, and was negatively related to the carbon (C) use efficiency of alanine, suggesting that microbial demand for C could partially drive the assimilation of soil organic N. These results suggest that the successional stage of tree species play an important role in regulating the turnover rates of soil organic N. An improved understanding of how tree species from different successional stages influence microbial function and soil organic N cycling is beneficial to future afforestation and forest management, alleviating the impacts of global change on the ecosystem.

SLC38A2 provides proline and alanine to regulate postnatal bone mass accrual in mice.

Amino acids have recently emerged as important regulators of osteoblast differentiation and bone formation. Osteoblasts require a continuous supply of amino acids to sustain biomass production to fuel cell proliferation, osteoblast differentiation and bone matrix production. We recently identified proline as an essential amino acid for bone development by fulfilling unique synthetic demands that are associated with osteoblast differentiation. Osteoblasts rely on the amino acid transporter SLC38A2 to provide proline to fuel endochondral ossification. Despite this, very little is known about the function or substrates of SLC38A2 during bone homeostasis. Here we demonstrate that the neutral amino acid transporter SLC38A2 is expressed in osteoblast lineage cells and provides proline and alanine to osteoblast lineage cells. Genetic ablation of SLC38A2 using Prrx1Cre results in decreased bone mass in both male and female mice due to a reduction in osteoblast numbers and bone forming activity. Decreased osteoblast numbers are attributed to impaired proliferation and osteogenic differentiation of skeletal stem and progenitor cells. Collectively, these data highlight the necessity of SLC38A2-mediated proline and alanine uptake during postnatal bone formation and bone homeostasis.

MRI measurement of alanine uptake in a mouse xenograft model of U-87 MG glioblastoma

The potential use of alanine as an MRI contrast agent was investigated. The relaxation properties of alanine solutions were measured at 9.4 T. The T2 relaxivity caused by the chemical exchange (R2ex) between amine protons and water protons was 0.10 mM−1 s−1 at 37 °C. As a demonstration, alanine uptake in a mouse xenograft model of U-87 MG glioblastoma was measured using MRI, and was compared with immunohistochemistry staining of ASCT2, a transporter that imports amino acids into cancer cells. Statistically significant (p = 0.0079) differences in ASCT2 distribution were found between regions that show strong and weak alanine uptake in MRI. To better understand the influence of perfusion, the effect of ASCT2 inhibition on the alanine uptake in MRI was investigated, and dynamic contrast enhanced MRI was compared with alanine MRI.

Abstract 469: A multi-scale analysis and visualization platform for cancer data - deriving tumor microenvironment behavior from pathology and transcriptomics

Abstract Heterogeneity is a hallmark of cancer and perhaps one of the most important features associated with resistance to therapies and likelihood of recurrence and/or metastasis. Genomic instability contributes to genetic diversity, which leads to high levels of intratumoural heterogeneity. While genetic diversity is one contributor to phenotypic heterogeneity in tumors, the spatial variation in the tumor microenvironment (TME) also drives emergent phenotypic heterogeneity and intrinsic variation in resistance to drugs and the immune system. Ultimately, heterogeneity leads to higher likelihood of metastasis. Traditionally, pathology allowed for the assessment of heterogeneity. However, this was limited to gross assessment of the TME, through measurement of mitotic levels and severity of cancer invasion. We present SimBioSys PhenoScope, a multi-scale analysis and visualization platform that integrates cancer data across scales to extract cross modality trends that drive cancer invasion. As a demonstrated use case for the platform, we present a vignette of using the platform to analyze pathology slides at three scales. Three convolutional neural networks (CNN) are developed and validated. The outputs of these networks were combined with 2D simulations of the metabolic behavior and growth of cells within the TME. Two CNNs were developed and one implemented: one that identifies cells undergoing mitosis, one that segments individual cells and classifies their type, and one that segments five tissues from pathology slides. Additionally, transcriptional data was used to generate patient specific metabolic models. Each CNN was developed using training images and validated on the test images. The mitosis detection CNN was found to have an accuracy of 76.2% (precision=83%, recall=76%) in the test set. The classification CNN was found to have a Dice Similarity Coefficient (DSC) of 0.821 for segmenting cells and an F1 score for classifying cells ranging from 0.559 (F1i) to 0.756 (F1d). The segmentation CNN was found to have an accuracy of 78.1% with DSC ranging from 0.66 and 0.86 depending on tissue. The segmentations were input into a proprietary simulation framework along with patient-specific metabolic models to predict the spatial gradients of nutrients, and spatial organization of growth and metabolism. Simulations show multiple behaviors such as regions of high lactate production or consumption by cancer, and regions that differ by lactate production and alanine uptake in cancers. These behaviors were correlated with local cell mitoses and invasion of Tumor Infiltrating Lymphocytes. Tools to examine cancers across scales and within the TME are currently lacking. We demonstrate a proof-of-principle approach of combining data across scales in a fashion that allows for novel predictions of TME behavior. Citation Format: Joseph R. Peterson, Matthew Shin, Patricia Carrigan, Michael J. Hallock, Snehal Patel, The SimBioSys Team. A multi-scale analysis and visualization platform for cancer data - deriving tumor microenvironment behavior from pathology and transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 469.

Nutrient availability regulates proline/alanine transporters in Trypanosoma brucei

Trypanosoma brucei is a species of unicellular parasite that can cause severe diseases in livestock and humans, including African trypanosomiasis and Chagas disease. Adaptation to diverse environments and changes in nutritional conditions is essential for T. brucei to establish an infection when changing hosts or during invasion of different host tissues. One such adaptation is the ability of T. brucei to rapidly switch its energy metabolism from glucose metabolism in the mammalian blood to proline catabolism in the insect stages and vice versa. However, the mechanisms that support the parasite's response to nutrient availability remain unclear. Using RNAseq and qRT-PCR, we investigated the response of T. brucei to amino acid or glucose starvation and found increased mRNA levels of several amino acid transporters, including all genes of the amino acid transporter AAT7-B subgroup. Functional characterization revealed that AAT7-B members are plasma membrane-localized in T. brucei and when expressed in Saccharomyces cerevisiae supported the uptake of proline, alanine, and cysteine, while other amino acids were poorly recognized. All AAT7-B members showed a preference for proline, which is transported with high or low affinity. RNAi-mediated AAT7-B downregulation resulted in a reduction of intracellular proline concentrations and growth arrest under low proline availability in cultured procyclic form parasites. Taken together, these results suggest a role of AAT7-B transporters in the response of T. brucei to proline starvation and proline catabolism.

Annexin A6 Is Critical to Maintain Glucose Homeostasis and Survival During Liver Regeneration in Mice.

Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48hours after PHx, impairing alanine uptake and, consequently, glucose production. We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.

Changes to hepatic nutrient dynamics and energetics in rainbow trout (Oncorhynchus mykiss) following exposure to and recovery from hydraulic fracturing flowback and produced water

Hydraulic fracturing flowback and produced water (FPW) is a highly complex and heterogenous wastewater by-product of hydraulic fracturing practices. To date, no research has examined how FPW exposure to freshwater biota may affect energetic homeostasis following subsequent induction of detoxification processes. Rainbow trout (Oncorhynchus mykiss) were acutely exposed for 48 h to either 2.5% or 7.5% FPW, and hepatic metabolism was assessed either immediately or following a 3-week recovery period. Induction of xenobiotic metabolism was observed with an 8.8-fold increase in ethoxyresorufin-O-deethylase (EROD) activity after 48 h exposure to 7.5% FPW, alongside a 10.3-fold increase in the mRNA abundance of cyp1a, both of which returned to basal level after three weeks. Glucose uptake capacity was elevated by 6.8- and 12.9-fold following 2.5% and 7.5% FPW exposure, respectively, while alanine uptake was variable. Activity measurements and mRNA abundance of key enzymes involved in hepatic metabolism indicated that aerobic metabolism was maintained with exposure, as was glycolysis. Gluconeogenesis, as measured by phosphoenolpyruvate carboxykinase (PEPCK) activity, decreased by ~30% 48 h following 2.5% FPW exposure and ~20% 3 weeks after 7.5% FPW exposure. The abundance of pepck mRNA activity followed similar, yet non-significant, trends. Finally, a delayed increase in amino acid catabolism was observed, as glutamate dehydrogenase (GDH) activity was increased 2-fold in 7.5% FPW exposed fish when compared to saline control fish at the 3-week time point. We provide evidence to suggest that although hepatic metabolism is altered following acute FPW exposure, metabolic homeostasis generally returns 3-weeks post-exposure.

Glutaminase Inhibition on NSCLC Depends on Extracellular Alanine Exploitation.

Non-small-cell lung cancer (NSCLC) cell lines vary in their sensitivity to glutaminase inhibitors, so it is important to identify the metabolic assets underling their efficacy in cancer cells. Even though specific genetic lesions such as in KRAS and LKB1 have been associated with reliance on glutamine for their metabolic needs, we found no distinction between glutaminase inhibitor CB-839 sensitivity and resistant phenotypes in NSCLC cells with or without these genetic alterations. We demonstrated the close relationship between environmental alanine uptake and catabolism. This response depended on the individual cell’s ability to employ alanine aminotransferase (GPT2) to compensate the reduced glutamate availability. It may, therefore, be useful to determine GPT2 levels to predict which NSCLC patients would benefit most from glutaminase inhibitor treatment.

Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters. PSCs utilize SLC1A4 and other transporters to rapidly exchange and maintain environmental alanine concentrations. Moreover, PDAC cells upregulate SLC38A2 to supply their increased alanine demand. Cells lacking SLC38A2 fail to concentrate intracellular alanine and undergo a profound metabolic crisis resulting in markedly impaired tumor growth. Our results demonstrate that stromal-cancer metabolic niches can form through differential transporter expression, creating unique therapeutic opportunities to target metabolic demands of cancer. SIGNIFICANCE: This work identifies critical neutral amino acid transporters involved in channeling alanine between pancreatic stellate and PDAC cells. Targeting PDAC-specific alanine uptake results in a metabolic crisis impairing metabolism, proliferation, and tumor growth. PDAC cells specifically activate and require SLC38A2 to fuel their alanine demands that may be exploited therapeutically.This article is highlighted in the In This Issue feature, p. 890.

Experimental strategies to measure the microbial uptake and mineralization kinetics of dissolved organic carbon in soil

Soil organic matter turnover rates are typically estimated from mass loss of the material over time or from on rates of carbon dioxide production. In the study, we investigated a new way to characterize the concentration-dependent kinetics of amino acids used by measuring microbial uptake and mineralization of 14C-alanine. We measured the depletion from soil solution after additions 14C-alanine. The microbial uptake of 14C-alanine from soil solution was concentration-dependent and kinetic analysis indicated the operation of at least three distinct alanine transport systems of differing affinities. Most of the 14C-alanine depletion from the soil solution occurred rapidly within the first 10–30 min of the incubation after 10 µM to 1 mM substrate additions. At alanine concentrations less than 250 mM, the kinetic parameters for Km and Vmax of the higher-affinity transporter were 60.0 µM and 1.32 µmol g−1 DW soil h−1, respectively. The mineralization of alanine was determined and the half-time values for the rapid mineralization process were 45 min to 1.5 h after the addition at alanine concentrations below 1 mM. The time delay after its uptake into microbial biomass suggested that alanine uptake and subsequent respiration were uncoupled pattern. The microbial N uptake rate was calculated by microbial mineralization, and an estimated Km value of 1731.7±274.6 µM and Vmax value of 486.0±38.5 µmol kg−1 DW soil h−1. This study provides an alternative approach for measuring the rate of turnover of compounds that turnover very rapidly in soil.

Expression study of an Amino Acid Permease-like gene in Phaseolus vulgaris L.

Amino acid permease-like (AAP-like) gene plays a critical role in absorbing amino acids through roots in plants. A number of studies have been done on amino acids uptake in plants but till date there is no report about the expression of AAP gene in Phaseolus under field allied condition. The aim of this study is to measure the expression of AAP-like gene on alanine, glycine and proline amino acid uptake capacity in Phaseolus vulgaris at field relevant concentrations. Amongst three amino acids, a drastic significant increase of 63.15 fold in expression of AAP-like gene is observed in 50 µM alanine at 2 hr. At 50 µM of proline and 25 µM of alanine, AAP-like gene expression also shows high expression of 43.71 fold at 2 hr and 42.50 fold at 1 hr respectively. This study elucidated the dose dependent relationship of glycine, alanine and proline with the expression of AAP-like gene in amino acid transport in natural conditions in roots of P. vulgaris. Additionally, this research is also useful in identification of plants needing less surplus nitrogen additions and helpful in optimizing fertilizers by tailoring AAP gene expression to match plant uptake capacities in agriculture.

Sensing Living Bacteria in Vivo Using d-Alanine-Derived 11C Radiotracers.

Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-11C]Alanine and the dipeptide d-[3-11C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [11C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-11C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[18F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-11C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-11C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-11C]alanine to address a number of important human infections.

Acquisition of alanyl-alanine in an Agnathan: Characteristics of dipeptide transport across the hindgut of the Pacific hagfish Eptatretus stoutii.

This study used 3 H-L -alanyl-L -alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (Jmax -like) of 70 ± 3 nmol cm-2 h-1 and an affinity constant (Km -like) of 1072 ± 81 μM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a Km distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L -alanyl-L -alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals.

Correction for Girard et al., "Uptake of l-Alanine and Its Distinct Roles in the Bioenergetics of Trypanosoma cruzi".

Volume 3, no. 4, e00338-18, 2018, [https://doi.org/10.1128/mSphereDirect.00338-18][1]. The following is not a correction per se but rather a clarification of some points in our published article. We calculated the kinetic and thermodynamic properties of the alanine uptake system in Trypanosoma cruzi

Plant-microbe competition: does injection of isotopes of C and N into the rhizosphere effectively characterise plant use of soil N?

Despite considerable attention over the last 25yr, the importance of early protein breakdown products to plant nitrogen (N) nutrition remains uncertain. We used rhizosphere injection of 15 N-, 13 C- and 14 C-labelled inorganic N and amino acid (l-alanine), with chase periods from 1min to 24h, to investigate the duration of competition for amino acid between roots (Triticum aestivum) and soil microorganisms. We further investigated how microbial modification of l-alanine influenced plant carbon (C) and N recovery. From recovery of C isotopes, intact alanine uptake was 0.2-1.3% of added. Soil microbes appeared to remove alanine from soil solution within 1min and release enough NH4+ to account for all plant 15 N recovery (over 24h) within 5min. Microbially generated inorganic or keto acid C accounted for <25% of the lowest estimate of intact alanine uptake. Co-location of C and N labels appears a reasonable measure of intact uptake. Potential interference from microbially modified C is probably modest, but may increase with chase period. Similarly, competition for l-alanine is complete within a few minutes in soil, whereas NO3- added at the same rate is available for >24h, indicating that long chase periods bias outcomes and fail to accurately simulate soil processes.

Warming alters competition for organic and inorganic nitrogen between co-existing grassland plant species

Grass species may acquire different forms of nitrogen (N) to reduce competition for the same resources. Climate change influences the availability of soil N and is therefore likely to cause shifts in N forms acquired by plants, thereby affecting their competitive interactions. We investigated the effects of warming on the uptake of different N forms and competitive interactions of Festuca ovina and Anthoxanthum odoratum in a pot experiment. The plants were grown either in monocultures or mixture, and at ambient or elevated temperature (+10 °C), and supplied with 13C and 15N isotopes to test for treatment effects on the relative uptake of ammonium, alanine or tri-alanine. Both grass species took up relatively more N supplied as ammonium than as alanine or tri-alanine when grown under ambient conditions in monoculture. In contrast, when grown in mixtures, F. ovina took up the three supplied N forms in equal amounts, whereas A. odoratum switched to tri-alanine as the main N form. Under warmed conditions, both species took up the N forms equally, irrespective of competition treatments. We have shown that grass species grown in mixture and under ambient conditions reduce competition by acquiring different N forms. Warming increased the availability of inorganic N in the soil and therefore deregulated the need for differential uptake of N forms.