Amino Acid Supply Research Articles

Stabilization of single-chain Fv antibody production using recombinant Escherichia coli by DO-stat fed-batch culture employing yeast extract-enriched feeding medium

The stabilized production of anti-CRP single-chain Fv (scFv) antibody using recombinant E. coli by prolonged dissolved oxygen (DO)-stat fed-batch culture was demonstrated, focusing on the supply of nitrogen sources during fed-batch operation. The serious degradation of anti-CRP scFv by intracellular proteases was observed when the fermentation time was prolonged from 50 h to 100 h, which was presumably induced by the insufficient supply of free amino acids. To evaluate the effects of amino acid supply on the proteolysis of anti-CRP scFv, fed-batch experiments using yeast extract (YE)-enriched feeding medium, which had various blend ratios of glucose and YE (G/YE), were performed. By increasing the YE content in the feeding medium, the expression of intracellular proteases was significantly suppressed; consequently, the proteolysis of the scFv production was successfully prevented in 100 h fed-batch culture. The desired G/YE ratio to avoid proteolysis was 1.0 g-glucose/g-YE. The stabilization of scFv production accordingly resulted in the enhanced scFv production from 3.0 g/L in 50 h fed-batch culture to 4.0–5.2 g/L in 100 h fed-batch culture with more than 97% solubility which corresponded to 94.9% of increase in total scFv production by one fed-batch operation.

Beneficial Effects of Soybean-Derived Bioactive Peptides.

Peptides present in foods are involved in nutritional functions by supplying amino acids; sensory functions related to taste or solubility, emulsification, etc.; and bioregulatory functions in various physiological activities. In particular, peptides have a wide range of physiological functions, including as anticancer agents and in lowering blood pressure and serum cholesterol levels, enhancing immunity, and promoting calcium absorption. Soy protein can be partially hydrolyzed enzymatically to physiologically active soy (or soybean) peptides (SPs), which not only exert physiological functions but also help amino acid absorption in the body and reduce bitterness by hydrolyzing hydrophobic amino acids from the C- or N-terminus of soy proteins. They also possess significant gel-forming, emulsifying, and foaming abilities. SPs are expected to be able to prevent and treat atherosclerosis by inhibiting the reabsorption of bile acids in the digestive system, thereby reducing blood cholesterol, low-density lipoprotein, and fat levels. In addition, soy contains blood pressure-lowering peptides that inhibit angiotensin-I converting enzyme activity and antithrombotic peptides that inhibit platelet aggregation, as well as anticancer, antioxidative, antimicrobial, immunoregulatory, opiate-like, hypocholesterolemic, and antihypertensive activities. In animal models, neuroprotective and cognitive capacity as well as cardiovascular activity have been reported. SPs also inhibit chronic kidney disease and tumor cell growth by regulating the expression of genes associated with apoptosis, inflammation, cell cycle arrest, invasion, and metastasis. Recently, various functions of soybeans, including their physiologically active functions, have been applied to health-oriented foods, functional foods, pharmaceuticals, and cosmetics. This review introduces some current results on the role of bioactive peptides found in soybeans related to health functions.

Assessment of vegan complete diets for dogs and cats available in Germany

Nutritional assessment of commercial vegan complete diets for adult cats and dogs and the evaluation of the products' legal labelling. Four complete vegan diets for adult cats and dogs available on the German market were analyzed concerning their nutrient and mineral content. The required amount of food was determined by calculation and compared to the manufacturers' feeding recommendation. Data labelling was assessed for its accordance with EC regulation 767/2009. The supply of taurine and vitamins was evaluated for its adequacy by use of the nutritional additive data on the product label. In most cases, the labelled data complied with the regulatory framework. The manufacturers' feeding recommendation differed significantly from the calculated feed quantity in 2 out of 4 feeds. The recommendations resulted in a coverage of 64 to 121 % of the total energy requirement, more frequently leading to an underestimation of the required feed amount. The supply of minerals exceeded the recommended allowance by 5.2- to 8.2-fold in the adult dog and by 9.0- and 11.4-fold in the adult cat. In 1 out of 4 diets, the European Pet Food Industry Federation guidelines were undercut for crude protein, crude fat and phosphorus. Zinc and copper contents were in excess and/or insufficient. A shortfall in National Research Council (NRC) recommendations was present for zinc content in one diet. Most products met the recommended supply of essential amino acids; 2 products only met the minimum requirements for methionine and cystine but not the recommended provision. The added taurine met the recommended intake in one commercial diet for cats. Requirements for vitamin intake were fulfilled by the nutritional additives in 3 products. None of the products fulfilled the energy and nutrient requirements for a complete diet for adult dogs and cats without limitations. Therefore, deficiencies in the supply of individual nutrients cannot be ruled out under long-term feeding of the examined vegan diets.

Assessing growth, frost tolerance, and acclimation of pine seedlings with contrasted dormancy strategies as influenced by organic nitrogen supply.

Freezing stress is a critical environmental factor affecting survival, distribution, and evolution of plants. Although there is evidence that nitrogen (N) affects frost tolerance of juvenile conifers, the magnitude and direction of such effect can diverge among species. The influence of the N source on frost tolerance has been barely studied. Particularly, how organic N sources could affect the cold acclimation dynamics of seedlings is poorly understood. We studied morpho-physiological responses to organic N supply (amino acids) in comparison to inorganic N in seedlings of two Mediterranean pine species: Pinus halepensis and P. sylvestris. Fertilization was applied at low and high N doses (30 and 130 mg N seedling-1 ) in the first growing season. Then, tolerance of seedlings to freezing stress was evaluated through the cold season. This study confirmed that organic N supply promotes growth of both species as effectively as inorganic N sources. At low N availability, seedlings had acute phosphorus deficiencies when grown with inorganic N, but not with organic N. Likewise, high organic-N availability improved chlorophylls concentration. Both species increased their frost tolerance through time, especially during late autumn. Although organic N supply did not show clear benefits on frost tolerance, it seemed to enhance cold acclimation via increases of compatible solutes, such as soluble sugars and proline, particularly in P. halepensis. Thus, the effects of organic N supply could depend on the extent that such osmolytes contribute to the dormancy strategy of the species. Other species-specific mechanisms to cope with freezing stress are further discussed.

Etiology of Colitis-Complex Diarrhea in Growing Pigs: A Review

Simple SummaryDiarrhea in growing pigs is a challenge for the pig industry since it is associated with reduced animal welfare, retarded growth, increased feed conversion ratio, and is often treated with antibiotics. One of the major causes of diarrhea in the growing period is large intestinal inflammation, often referred to as colitis. The exact causes of colitis-complex diarrhea are still to be understood, but dietary factors and/or pathogens have been recognized as the major factors in developing colitis-complex diarrhea. In this review, a thorough picture of pathogens, dietary factors, and a number of possible biomarkers related to colitis-complex diarrhea is presented.Colitis-complex diarrhea (CCD) in pigs can be defined as a type of diarrhea, which is associated with colonic inflammation and disrupted colonic gut barrier functionality in growing pigs (4–16 weeks post-weaning). It is a challenge for the pig industry as it is associated with the high use of antibiotics, reduced animal welfare, and depressed growth rate. The exact etiology of CCD is still unclear; however, pathogens including Brachyspira (B.) hyodysenteriae, B. pilosicoli, and swine whipworms such as Trichuris (T.) suis have been involved in specific colitis (SC). In the absence of specific pathogens, dietary factors, such as high levels of protein, pelleted feedstuffs, and lack of sufficient antioxidants, can result in non-specific colitis (NSC). On the other hand, supplement of polyunsaturated fatty acids (PUFA) and polyphenols, sufficient supply of essential amino acids (e.g., threonine, cysteine, and proline), short-chain fatty acids (SCFA; especially butyrate), and resistant starch have shown to confer preventing/ameliorating effects on CCD. Different putative biomarkers associated with CCD have been presented. It is anticipated that a comprehensive picture of the possible causes of CCD and potential dietary interventions could cast light on the direction of future studies aimed at developing preventive and curative strategies against CCD in growing pigs.

Amino Acids Supplementation for the Milk and Milk Protein Production of Dairy Cows.

Simple SummaryThe composition of milk not only has nutritional implications, but is also directly related to the income of dairy producers. As regards milk’s composition, concerns around milk protein have emerged from the increased consumption of casein products. The synthesis of proteins in milk is a highly complex and high-cost process, because the conversion efficiency of dietary protein to milk protein is very low in dairy cows. Thus, some studies have increased milk protein by using protein supplements or a single amino acid (AA) supply. AAs are the building blocks of protein, and can also stimulate the protein synthetic pathway. This review mainly concerns the use of AAs for producing milk protein in high-producing dairy cows, particularly with methionine, lysine, and histidine. Understanding the mechanisms of AAs will help to promote milk protein synthesis in the dairy industry.As the preference of consumers for casein products has increased, the protein content of milk from dairy cows is drawing more attention. Protein synthesis in the milk of dairy cows requires a proper supply of dietary protein. High protein supplementation may help to produce more milk protein, but residues in feces and urine cause environmental pollution and increase production costs. As such, previous studies have focused on protein supplements and amino acid (AA) supply. This review concerns AA nutrition for enhancing milk protein in dairy cows, and mainly focuses on three AAs: methionine, lysine, and histidine. AA supplementation for promoting protein synthesis is related to the mammalian target of rapamycin (mTOR) complex and its downstream pathways. Each AA has different stimulating effects on the mTOR translation initiation pathway, and thus manifests different milk protein yields. This review will expand our understanding of AA nutrition and the involved pathways in relation to the synthesis of milk protein in dairy cows.

Robustness against point mutations of genetic code extensions under consideration of wobble-like effects

Many theories of the evolution of the genetic code assume that the genetic code has always evolved in the direction of increasing the supply of amino acids to be encoded (Barbieri, 2019; Di Giulio, 2005; Wong, 1975). In order to reduce the risk of the formation of a non-functional protein due to point mutations, nature is said to have built in control mechanisms. Using graph theory the authors have investigated in Blazej et al. (2019) if this robustness is optimal in the sense that a different codon–amino acid assignment would not generate a code that is even more robust. At present, efforts to expand the genetic code are very relevant in biotechnological applications, for example, for the synthesis of new drugs (Anderson et al., 2004; Chin, 2017; Dien et al., 2018; Kimoto et al., 2009; Neumann et al., 2010). In this paper we generalize the approach proposed in Blazej et al. (2019) and will explore hypothetical extensions of the standard genetic code with respect to their optimal robustness in two ways:(1) We keep the usual genetic alphabet but move from codons to longer words, such as tetranucleotides. This increases the supply of coding words and thus makes it possible to encode non-canonical amino acids.(2) We expand the genetic alphabet by introducing non-canonical base pairs. In addition, the approach from Blazej et al. (2019) and Blazej et al. (2018) is extended by incorporating the weights of single point-mutations into the model. The weights can be interpreted as probabilities (appropriately normalized) or degrees of severity of a single point mutation. In particular, this new approach allows us to take a closer look at the wobble effects in the translation of codons into amino acids. According to the results from Blazej et al. (2019) and Blazej et al. (2018), the standard genetic code is not optimal in terms of its robustness to point mutations if the weights of single point mutations are not taken into account. After incorporation into the model weights that mimic the wobble effect, the results of the present work show that it is much more robust, almost optimal in that respect.We hope, that this theoretical analysis might help to assess extended genetic codes and their abilities to encode new amino acids.

Impact of lysine supplementation on growth and development ofHermetia illucens larvae

Hermetia illucens larvae are able to convert a broad range of organic substrates into animal protein and are believed to have the potential to help solving the supply problem for animal protein in the future. Many farmed animals such as poultry or pigs need an adequate amount of essential amino acids like lysine for optimal growth and performance. In most diets for farmed animals, essential amino acids are supplemented as feed additive to avoid excessive protein intake while ensuring a sufficient supply of essential amino acids. There is no information on the requirements of lysine for the growth ofH. illucens larvae. Furthermore, it remains open whether lysine may have an extra effect on growth and development in the cultivation of such larvae. The aim of the study was to evaluate, if an addition of lysine to their feed substrate will affect these parameters. Therefore, in the present study up to 3.0% crystalline lysine was added to a basic substrate and survival, growth, and development of larvae were recorded. Furthermore, larval nutritional values like crude fat, crude protein, and lysine content were analysed. The addition of lysine did not improve growth, development or nutritional values ofH. illucens larvae. In contrast, the addition of lysine led to reduced larval size, survival and prepupal rates, especially when high amounts of lysine were provided. The results of this study indicate that a lysine content of 0.3% in the substrate is sufficient for optimal larval growth. UsingH. illucens larvae for animal protein production could, therefore, be accomplished with less lysine compared to many other farmed animals.

Oncogene-regulated release of extracellular vesicles.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

Acinetobacter tandoii ZM06 Assists Glutamicibacter nicotianae ZM05 in Resisting Cadmium Pressure to Preserve Dipropyl Phthalate Biodegradation.

Dipropyl phthalate (DPrP) coexists with cadmium as cocontaminants in environmental media. A coculture system including the DPrP-degrading bacterium Glutamicibacter nicotianae ZM05 and the nondegrading bacterium Acinetobacter tandoii ZM06 was artificially established to degrade DPrP under Cd(II) stress. Strain ZM06 relieved the pressure of cadmium on strain ZM05 and accelerated DPrP degradation in the following three ways: first, strain ZM06 adsorbed Cd(II) on the cell surface (as observed by scanning electron microscopy) to decrease the concentration of Cd(II) in the coculture system; second, the downstream metabolites of ZM05 were utilized by strain ZM06 to reduce metabolite inhibition; and third, strain ZM06 supplied amino acids and fatty acids to strain ZM05 to relieve stress during DPrP degradation, which was demonstrated by comparative transcriptomic analysis. This study provides an elementary understanding of how microbial consortia improve the degradation efficiency of organic pollutants under heavy metals contamination.

Why is phloem sap nitrate kept low?

Since the first description of phloem sap composition nearly 60 years ago, it is generally assumed that phloem sap does not contain nitrate and that there is little or no backflow of nitrate from shoots to roots. While it is true that nitrate can occasionally be absent from phloem sap, there is now substantial evidence that phloem can carry nitrate and furthermore, transporters involved in nitrate redistribution to shoot sink organs and roots have been found. This raises the question of why nitrate may or may not be present in phloem sap, why its concentration is generally kept low, and whether plant shoot-root nutrient cycling also involves nitrate. We propose here that phloem sap nitrate is not only an essential component of plant nutritional signaling but also contributes to physical properties of phloem sap and as such, its concentration must be controlled to ensure proper coordination of plant development and nutrient transport. This article is protected by copyright. All rights reserved.

Ethnobotanical survey, phytoconstituents and antibacterial investigation of Rapanea melanophloeos (L.) Mez. bark, fruit and leaf extracts

Abstract Rapanea melanophloeos is traditionally used in South Africa in the treatment of ailments of the skin, pulmonary and gastro intestinal tract. This study was aimed at giving an overview of these traditional uses and comparing the phytochemicals and antibacterial activities of various crude extracts of the leaves, fruits and bark in order to validate these uses. The three plant parts were extracted using petroleum ether (PE), ethyl acetate (EtOAc), methanol (MeOH) and water. Various phytochemicals were compared using TLC, while alcohol precipitable solids (APS), non-polar terpenes and amino acids were analysed by GC-MS. Antibacterial activity was determined against three Gram-positive and three Gram-negative strains by microdilution assays. Caryophyllene oxides, α-cadinol and (−)-spathulenol were identified in the PE extracts. All nine essential amino acids were present in fruit extracts in significantly higher levels than in the leaves and bark; 255.1, 23.4 and 21.3 mg/g respectively. Most of the extracts showed good antibacterial activity, especially against the Gram-positive pathogens (MIC of ≤1 mg/mL), the EtOAc extracts exhibited the best activity with the fruit having an MIC values of 0.1 ± 0.2 mg/mL against Staphylococcus epidermidis and Enterococcus faecalis, 0.05 mg/mL against Bacillus cereus. Results from this study validate the ethnomedicinal uses of R. melanophloeos extracts for ailments of bacterial etiology. The plant had a rich supply of secondary metabolites, APS and amino acids and TLC and antibacterial activities of the extracts showed slight variations in chemical composition due to geographic distribution.

Population protein intakes and food sustainability indices: The metrics matter

It is common practice in food sustainability studies to calculate population-based average protein intakes and express environmental footprints on a gross protein basis. Such analyses, however, assume that the requirement for protein is also expressed as gross protein which is not the case. This ignores the critical role of dietary protein as a supply of dietary indispensable amino acids, and does not account for differences in the protein quality of foods.Previously published datasets are re-analysed here to express population protein intakes and environmental footprint data on an available protein basis, accounting for both amino acid digestibility and utilization. When protein quality is taken into account, for many countries of the world protein is found to be deficient for the average adult, and different rankings are found for various environmental footprints.

Singapore multidisciplinary consensus recommendations on muscle health in older adults: assessment and multimodal targeted intervention across the continuum of care

BackgroundThe rapidly aging societies worldwide and in Singapore present a unique challenge, requiring an integrated multidisciplinary approach to address high-value targets such as muscle health. We propose pragmatic evidence-based multidisciplinary consensus recommendations for the assessment and multi-modal management of muscle health in older adults (≥65 years) across the continuum of care.MethodsThe recommendations are derived from an in-depth review of published literature by a multidisciplinary working group with clinical experience in the care of the older population in both acute and community settings.ResultsThe panel recommends screening for muscle impairment using the SARC-F questionnaire, followed by assessment for low muscle strength (handgrip strength or 5-times chair stand test ≥10 s as a surrogate for lower limb strength) to diagnose possible/probable sarcopenia. For uncomplicated cases, lifestyle modifications in exercise and diet can be initiated in the community setting without further assessment. Where indicated, individuals diagnosed with possible/probable sarcopenia should undergo further assessment.Diagnosis of sarcopenia should be based on low muscle strength and low muscle mass (bioimpedance analysis, dual-energy X-ray absorptiometry or calf circumference as a surrogate). The severity of sarcopenia should be determined by assessment of physical performance (gait speed or 5-times chair stand test ≥12 s as a surrogate for gait speed). To treat sarcopenia, we recommend a combination of progressive resistance-based exercise training and optimization of nutritional intake (energy, protein and functional ingredients). High quality protein in sufficient quantity, to overcome anabolic resistance in older adults, and distributed throughout the day to enable maximum muscle protein synthesis, is essential. The addition of resistance-based exercise training is synergistic in improving the sensitivity of muscle protein synthesis response to the provision of amino acids and reducing anabolic resistance. An expected dose-response relationship between the intensity of resistance-based training, lean mass and muscle strength is described.ConclusionsReviewed and endorsed by the Society of Rehabilitation Medicine Singapore and the Singapore Nutrition and Dietetics Association, these multidisciplinary consensus recommendations can provide guidance in the formulation of comprehensive and pragmatic management plans to improve muscle health in older adults in Singapore and Asia.

Enhanced small neutral but not branched chain amino acid transport after epigenetic sodium coupled neutral amino acid transporter-2 (SNAT2) cDNA expression in myoblasts.

BackgroundSkeletal muscle mass and function are partly maintained by the supply of amino acids, altered amino acid transport is an important cause of frailty that can lead to decreased independence with increasing age and slow trauma recovery. The system‐A sodium coupled neutral amino acid transporter (SNAT)‐2 coded by gene family SLC38A2 generates a 506 amino acid 56 kDa protein that is an important transporter of amino acids in skeletal muscle. Ageing is associated with a decrease in expression of SNAT2 transporters.MethodsIn this study, we used the C2C12 cell line, using myoblast cells and cells differentiated into myotubes. We investigated if the expression of SNAT2 DNA would enhance intracellular amino acid levels and increase their availability for protein synthesis.ResultsIn control myoblasts and myotubes, we found significantly decreased expression of SNAT2 (6.5× decrease, n = 4 per group, P < 0.05) in myotubes than found in myoblasts. After transfection with a SNAT2‐eGFP cDNA plasmid, C2C12 myoblasts significantly increased perinuclear punctate SNAT2‐eGFP expression that persisted and was more cytoplasmic after differentiation into myotubes. Interestingly, transfected cells were significantly more responsive to the hormone 5α‐dihydrotestosterone (DHT, 4.5 nM, by 1.6×, n = 3 per group, P < 0.04). Starvation significantly enhanced the amino acid C14‐MeAIB transport (1.7×, n = 3 per group, P < 0.05) indicating increased function of SNAT2. Inhibiting SNAT2 with high concentrations of MeAIB (3.3 or 5 mM) significantly reduced C14‐Isoleucine transport by L‐type amino acid transporter (LAT2, 52.8% and 77%, respectively, n = 3 per group, P < 0.05). However, there was no increase in the LAT2 transport of C14‐isoleucine detectable in SNAT2‐eGFP transfected cells after DHT (4.5 nM) exposure. This indicated that small amino acid availability was not rate limiting to LAT2 function in myoblasts.ConclusionsOverall, these data show that transfection of SNAT2‐eGFP expression enhanced its function following starvation and treatment with physiological levels of DHT. Enhanced SNAT2 expression in muscle cells offers a viable epigenetic target in pathological conditions associated with altered amino acid transport.

Counter-directed leucine gradient promotes amino acid transfer across the human placenta

The developing fetus is highly vulnerable to imbalances in the supply of essential amino acids (AA). Transplacental AA transfer depends on complex interactions between accumulative transporters, exchangers and facilitators, which maintain both intra-extracellular and materno-fetal substrate gradients. We determined physiological AA gradients between maternal and fetal blood and assessed their importance by studying maternal-fetal leucine transfer in human trophoblasts. Maternal-venous and corresponding fetal-arterial/fetal-venous sera were collected from 22 healthy patients at partum. The acquisition of the full AA spectra in serum was performed by ion exchange chromatography. Physiological materno-fetal AA levels were evaluated using paired two-way ANOVA with Tukey's correction. AA concentrations and gradients were tested for associations with anthropometric data by Spearman correlation analysis. Functional effects of a physiological leucine gradient versus equimolar concentrations were tested in BeWo cells using L-[3H]-leucine in conventional and Transwell-based uptake and transfer experiments. The LAT1/SLC7A5-specific inhibitor JPH203 was used to evaluate LAT1-transporter-mediated leucine transport. Maternal AA concentrations correlated with preconceptional and maternal weights at partum. Interestingly, low materno-fetal AA gradients were associated with maternal weight, BMI and gestational weight gain. Leucine uptake was promoted by increased extracellular substrate concentrations. Materno-fetal leucine transfer was significantly increased against a 137µM leucine gradient demonstrating that transplacental leucine transport is stimulated by a counter-directed gradient. Moreover, leucine transfer was inhibited by 10µM JPH203 confirming that Leu transport across the trophoblast monolayer is LAT1-dependent. This study demonstrates a currently underestimated effect of transplacental AA gradients on efficient leucine transfer which could severely affect fetal development.

Dietary Essential Amino Acid Restriction Promotes Hyperdipsia via Hepatic FGF21.

Prior studies have reported that dietary protein dilution (DPD) or amino acid dilution promotes heightened water intake (i.e., hyperdipsia) however, the exact dietary requirements and the mechanism responsible for this effect are still unknown. Here, we show that dietary amino acid (AA) restriction is sufficient and required to drive hyperdipsia during DPD. Our studies demonstrate that particularly dietary essential AA (EAA) restriction, but not non-EAA, is responsible for the hyperdipsic effect of total dietary AA restriction (DAR). Additionally, by using diets with varying amounts of individual EAA under constant total AA supply, we demonstrate that restriction of threonine (Thr) or tryptophan (Trp) is mandatory and sufficient for the effects of DAR on hyperdipsia and that liver-derived fibroblast growth factor 21 (FGF21) is required for this hyperdipsic effect. Strikingly, artificially introducing Thr de novo biosynthesis in hepatocytes reversed hyperdipsia during DAR. In summary, our results show that the DPD effects on hyperdipsia are induced by the deprivation of Thr and Trp, and in turn, via liver/hepatocyte-derived FGF21.

Molecular characteristics supporting l-Type amino acid transporter 1 (LAT1)-mediated translocation

l-Type amino acid transporter 1 (LAT1) is an interesting protein due to its peculiar expression profile. It can be utilized not only as a carrier for improved or targeted drug delivery, e.g., into the brain but also as a target protein by which amino acid supply can be restricted, e.g., from the cancer cells. The recognition and binding processes of LAT1-ligands, such as amino acids and clinically used small molecules, including l-dopa, gabapentin, and melphalan, are today well-known. Binding to LAT1 is crucial, particularly when designing the LAT1-inhibitors. However, it will not guarantee effective translocation across the cell membrane via LAT1, which is a definite requirement for LAT1-substrates, such as drugs that elicit their pharmacological effects inside the cells. Therefore, in the present study, the accumulation of known LAT1-utilizing compounds into the selected LAT1-expressing cancer cells (MCF-7) was explored experimentally over a time period. The differences found among the transport efficiency and affinity of the studied compounds for LAT1 were subsequently explained by docking the ligands into the human LAT1 model (based on the recent cryo-electron microscopy structure). Thus, the findings of this study clarify the favorable structural requirements of the size, shape, and polarity of the ligands that support the translocation and effective transport across the cell membrane via LAT1. This knowledge can be applied in future drug design to attain improved or targeted drug delivery and hence, successful LAT1-utilizing drugs with increased therapeutic effects.

Amino acids dominate diffusive nitrogen fluxes across soil depths in acidic tussock tundra.

Organic nitrogen (N) is abundant in soils, but early conceptual frameworks considered it nonessential for plant growth. It is now well recognised that plants have the potential to take up organic N. However, it is still unclear whether plants supplement their N requirements by taking up organic N insitu: at what rate is organic N diffusing towards roots and are plants taking it up? We combined microdialysis with live-root uptake experiments to measure amino acid speciation and diffusion rates towards roots of Eriophorum vaginatum. Amino acid diffusion rates (321ngNcm-2 h-1 ) were c. 3×higher than those for inorganic N. Positively charged amino acids made up 68% of the N diffusing through soils compared with neutral and negatively charged amino acids. Live-root uptake experiments confirmed that amino acids are taken up by plants (up to 1µgNg-1 min-1 potential net uptake). Amino acids must be considered when forecasting plant-available N, especially when they dominate the N supply, and when acidity favours proteolysis over net N mineralisation. Determining amino acid production pathways and supply rates will become increasingly important in projecting the extent and consequences of shrub expansion, especially considering the higher C:N ratio of plants relative to soil.

Supplementation of protease to low amino acid diets containing superdose level of phytase for wean-to-finish pigs: effects on performance, postweaning intestinal health and carcass characteristics.

This experiment investigated the effects of protease supplementation to low amino acid (AA) diets containing phytase on pig growth performance, postweaning intestinal health and carcass characteristics. A total of 936 weaned pigs (21 d of age, initial BW 5.87 ± 0.31 kg) were used in a 2 × 2 factorial design comparing the main effects of AA supply [standard feeding program: balanced for all nutrients with adjustment of Ca and P due to inclusion of phytase (2,500 FTU/kg in Phase 1 to 4; 500 FTU/kg in Phase 5 to 9) vs. low AA feeding program: 15% lower standardized ileal digestible lysine with relative reduction of all other essential AA] and protease level (0 vs. 0.0125%). Pens were assigned to dietary treatment according to a randomized complete block design with 26 pigs per pen and nine replicates per dietary treatment. Feed and water were provided on an ad libitum basis for all phases throughout the wean-to-finish period. Feed intake and body weight were determined every 2 wk during nursery period and monthly in the grow-finish period. Intestinal health in the first 17 d was assessed based on lactulose:mannitol ratio (L:M), serum IgA, and pen diarrhea assessment. Overall, pigs fed standard wean-to-finish diets had greater (P < 0.05) ADG and G:F than pigs fed low AA diets. Pig growth performance was not different throughout the wean-to-finish period with or without protease supplementation and with no interaction between AA supply and protease supplementation. There were no differences among dietary treatments for carcass characteristics. No difference was observed for urinary L:M and serum IgA; however, the L:M ratio was approximately 32% lower in pigs fed low AA diets + protease compared with pigs fed standard and low AA diets at d 5 and d 17 postweaning. Pigs fed protease supplemented diets had lower incidence of diarrhea (χ2 < 0.05) compared with pigs fed diets without protease. Results of the experiment indicate that dietary protease supplementation benefits intestinal health of nursery pigs.