Polar Metabolites Profiling of Wheat Shoots (Triticum aestivum L.) under Repeated Short-Term Soil Drought and Rewatering.

The response of wheat (Triticum aestivum L.) plants to the soil drought at the metabolome level is still not fully explained. In addition, research focuses mainly on single periods of drought, and there is still a lack of data on the response of plants to short-term cyclical periods of drought. The key to this research was to find out whether wheat shoots are able to resume metabolism after the stress subsides and if the reaction to subsequent stress is the same. Gas chromatography coupled with mass spectrometry (GC-MS) is one of the most valuable and fast methods to discover changes in the primary metabolism of plants. The targeted GC-MS analyses of whole shoots of wheat plants exposed (at the juvenile stage of development) to short-term (five days) mild soil drought/rewatering cycles (until the start of shoot wilting) enabled us to identify 32 polar metabolites. The obtained results revealed an accumulation of sugars (sucrose, fructose, glucose, and 1-kestose), proline, and malic acid. During five days of recovery, shoots regained full turgor and continued to grow, and the levels of accumulated metabolites decreased. Similar changes in metabolic profiles were found during the second drought/rewatering cycle. However, the concentrations of glucose, proline, and malic acid were higher after the second drought than after the first one. Additionally, the concentration of total polar metabolites after each plant rewatering was elevated compared to control samples. Although our results confirm the participation of proline in wheat responses to drought, they also highlight the responsiveness of soluble carbohydrate metabolism to stress/recovery.

Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells.

Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.

MxFRO4 confers iron and salt tolerance through up-regulating antioxidant capacity associated with the ROS scavenging.

Iron is involved in various metabolic pathways of plants. Stress from iron deficiency and toxicity in the soil adversely affects plant growth. Therefore, studying the mechanism of iron absorption and transport by plants is of important for resistance to iron stress and to increase crop yield. In this study, Malus xiaojinensis (a Fe-efficient Malus plant) was used as research material. A ferric reduction oxidase (FRO) family gene member was cloned and named MxFRO4. The MxFRO4 encoded a protein of 697 amino acid residues with a predicted molecular weight of 78.54 kDa and a theoretical isoelectric point of 4.90. A subcellular localization assay showed that the MxFRO4 protein was localized on the cell membrane. The expression of MxFRO4 was enriched in immature leaves and roots of M. xiaojinensis, and was strongly affected by low-iron, high-iron, and salt treatments. After introduction of MxFRO4 into Arabidopsis thaliana, the iron and salt stress tolerance of transgenic A. thaliana was greatly improved. Under exposure to low-iron and high-iron stresses, the primary root length, seedling fresh weight, contents of proline, chlorophyll, and iron, and iron(III) chelation activity of the transgenic lines were significantly increased compared with the wild type. The contents of chlorophyll and proline, and the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and catalase were significantly higher in transgenic A. thaliana overexpressing MxFRO4 under salt stress compared with the wild type, whereas the malondialdehyde content was decreased. These results suggest that MxFRO4 contributes to alleviating the effects of low-iron, high-iron, and salinity stresses in transgenic A. thaliana.

Synergistic n → π* and nN → π*Ar interactions in C-terminal modified prolines: effect on Xaa-Pro cis/trans equilibrium.

Carbonyl-carbonyl (CO⋯CO) n → π* interaction often coexists with a hydrogen bond (HB) or another n → π* interaction. Although the interplay between HB and n → π* interaction was previously studied, there is no systematic investigation that shows a synergistic relationship of n → π* with another noncovalent interaction. Herein, we have studied a set of proline-diacylhydrazine (Pro-DAH) molecules and observed that increase in the strength of the nN → π*Ar interaction on their DAH side strengthened the n → π* interaction on the Pro side, which was experimentally determined by measuring the Ktrans/cis of the Xaa-Pro amide bond. Overall, we describe a simple C-terminal modification strategy to stabilize the trans-Pro geometry that could be useful to stabilize PPII helices and collagen triple helices that require Pro to adopt the trans amide geometry.

Co-inoculation of Mycorrhiza and methyl jasmonate regulates morpho-physiological and antioxidant responses of Crocus sativus (Saffron) under salinity stress conditions.

Salinity stress is the second most devastating abiotic factor limiting plant growth and yields. Climate changes have significantly increased salinity levels of soil. Besides improving the physiological responses under stress conditions, jasmonates modulate Mycorrhiza-Plant relationships. The present study aimed to evaluate the effects of methyl jasmonate (MeJ) and Funneliformis mosseae (Arbuscular mycorrhizal (AM) on morphology and improving antioxidant mechanisms in Crocus sativus L. under salinity stress. After inoculation with AM, pre-treated C. sativus corms with MeJ were grown under low, moderate, and severe salinity stress. Intense salinity levels damaged the corm, root, total leaf dry weight, and area. Salinities up to 50 mM increased Proline content and Polyphenol oxidase (PPO) activity, but MeJ increased this trend in proline. Generally, MeJ increased anthocyanins, total soluble sugars, and PPO. Total chlorophyll and superoxide dismutase (SOD) activity increased by salinity. The maximum catalase and SOD activities in + MeJ + AM were 50 and 125 mM, respectively, and the maximum total chlorophyll in -MeJ + AM treatment was 75 mM. Although 20 and 50 mM increased plant growth, using mycorrhiza and jasmonate enhanced this trend. Moreover, these treatments reduced the damage of 75 and 100 mM salinity stress. Using MeJ and AM can improve the growth of saffron under various ranges of salinity stress levels; however, in severe levels like 120 mM, this phytohormone and F. mosseae effects on saffron could be adverse.

On the Presence of Gluten-Cleaving Activity in Sodiomyces alkalinus and S. magadiensis Strains.

Special enzymes are necessary for producing gluten-free foods, and specific proteolytic enzymes with gluten-degrading activity may be used as oral treatments for celiac disease. Enzymes of the kind were sought, identified, and preliminarily characterized in two strains of the alkaliphilic microscopic fungi Sodiomyces alkalinus and S. magadiensis. Post-glutamine cleaving activity was for the first time observed in the strains along with proline-cleaving activities of dipeptidyl peptidase 4 (DPP4) and proline aminopeptidase (PAP), allowing efficient hydrolysis of both proline/glutamine-rich gluten peptides and whole gluten. The optimal pH and pH-dependent stability were determined for the peptidases in question. All of the enzymes shown to cleave the prolyne/glutamine-containing bonds were assigned to the serine peptidase group and were found to be stable in moderately acidic and alkaline conditions. Owing to their activities, the peptidases are promising as tools to produce gluten-free foods and to design diets for gluten-intolerant patients.

Ultraviolet Photodissociation of Proteinogenic Amino Acids.

The ultraviolet photochemistry of the amino acids glycine, leucine, proline, and serine in their neutral forms was investigated using parahydrogen matrix-isolation spectroscopy. Irradiation by 213 nm light destroys the chirality of all three chiral amino acids as a result of the α-carbonyl C-C bond cleavage and hydrocarboxyl (HOCO) radical production. The temporal behavior of the Fourier-transform infrared spectra revealed that HOCO radicals rapidly reach a steady state, which occurs predominantly due to photodissociation of HOCO into CO + OH or CO2 + H. In glycine and leucine, the amine radicals generated by the α-carbonyl C-C bond cleavage rapidly undergo hydrogen elimination to yield methanimine and 3-methylbutane-1-imine, respectively. Breaking of the α-carbonyl C-C bond in proline appeared to yield 1-pyrroline, although due to its weak absorption it remains unconfirmed. In serine, additional products were formaldehyde and E/Z ethanimine. The present study shows that the direct production of HOCO previously observed in α-alanine generalizes to other amino acids of varying structure. It also revealed a tendency for amino acid photolysis to form imines rather than amine radicals. HOCO should be useful in the search for amino acids in interstellar space, particularly in combination with simple imine molecules.

Stachydrine, a Bioactive Equilibrist for Synephrine, Identified from Four Citrus Chinese Herbs.

Four Chinese herbs from the Citrus genus, namely Aurantii Fructus Immaturus (Zhishi), Aurantii Fructus (Zhiqiao), Citri Reticulatae Pericarpium Viride (Qingpi) and Citri Reticulatae Pericarpium (Chenpi), are widely used for treating various cardiovascular and gastrointestinal diseases. Many ingredients have already been identified from these herbs, and their various bioactivities provide some interpretations for the pharmacological functions of these herbs. However, the complex functions of these herbs imply undisclosed cholinergic activity. To discover some ingredients with cholinergic activity and further clarify possible reasons for the complex pharmacological functions presented by these herbs, depending on the extended structure-activity relationships of cholinergic and anti-cholinergic agents, a simple method was established here for quickly discovering possible choline analogs using a specific TLC method, and then stachydrine and choline were first identified from these Citrus herb decoctions based on their NMR and HRMS data. After this, two TLC scanning (TLCS) methods were first established for the quantitative analyses of stachydrine and choline, and the contents of the two ingredients and synephrine in 39 samples were determined using the valid TLCS and HPLC methods, respectively. The results showed that the contents of stachydrine (3.04‱) were 2.4 times greater than those of synephrine (1.25‱) in Zhiqiao and about one-third to two-thirds of those of Zhishi, Qingpi and Chenpi. Simultaneously, the contents of stachydrine, choline and synephrine in these herbs present similar decreasing trends with the delay of harvest time; e.g., those of stachydrine decrease from 5.16‱ (Zhishi) to 3.04‱ (Zhike) and from 1.98‱ (Qingpi) to 1.68‱ (Chenpi). Differently, the contents of synephrine decrease the fastest, while those of stachydrine decrease the slowest. Based on these results, compared with the pharmacological activities and pharmacokinetics reported for stachydrine and synephrine, it is indicated that stachydrine can be considered as a bioactive equilibrist for synephrine, especially in the cardio-cerebrovascular protection from these citrus herbs. Additionally, the results confirmed that stachydrine plays an important role in the pharmacological functions of these citrus herbs, especially in dual-directionally regulating the uterus, and in various beneficial effects on the cardio-cerebrovascular system, kidneys and liver.

Effects of elevated CO2 concentration on cell structure and stress resistance physiology of Setaria italica under drought stress.

The frequency of drought will increase under further warming. The increase in atmospheric CO2 concentration, along with more frequent drought, will affect crop growth. We examined the changes of cell structure, photosynthetic physiology, antioxidant enzymes, osmotic regulatory substances, and yield of foxtail millet (Setaria ita-lica) leaves under different CO2 concentrations (ambient air CO2 concentration and ambient atmospheric CO2 concentration + 200 µmol·mol-1) and water treatment (soil moisture content maintained at 45%-55%, and 70%-80% of field capacity, representing mild drought and normal water condition, respectively). The results showed that elevated CO2 concentration increased the number of starch grains, the area of single starch grains, and the total area of starch grains in the chloroplast of millet mesophyll cells. Under mild drought condition, elevated CO2 concentration increased net photosynthetic rate of millet leaves at the booting stage by 37.9%, but did not affect water use efficiency at this stage. Elevated CO2 concentration increased net photosynthetic rate and water use efficiency of millet leaves under mild drought condition at the filling stage by 15.0% and 44.2%, respectively. Under mild drought condition, elevated CO2 concentration increased the content of peroxidase (POD) and soluble sugar in millet leaves at the booting stage by 39.3% and 8.0%, respectively, but decreased proline content by 31.5%. It increased the content of POD in millet leaves at the filling stage by 26.5% but decreased the content of MDA and proline by 37.2% and 39.3%, respectively. Under mild drought condition, elevated CO2 concentration significantly increased the number of grain spikes by 44.7% and yield by 52.3% in both years compared with normal water condition. The effect of elevated CO2 concentration on grain yield under mild drought conditions was higher than that under normal water condition. Under mild drought conditions, elevated CO2 concentration increased leaf thickness, vascular bundle sheath cross-sectional area, net photosynthetic rate, and water use efficiency of millet, improved the antioxidant oxidase activity, and changed the concentration of osmotic regulatory substances, alleviated the nega-tive effect of drought on foxtail millet, and finally increased the number of grains per ear and yield of foxtail millet. This study would provide a theoretical basis for millet production and sustainable agricultural development in arid areas under future climate change.

Cerium and samarium blocked antioxidant enzymes in wheat plants.

This work was conducted to study positive and negative impacts of cerium (Ce) and samarium (Sm) on two cultivars (Arta and Baharan) in wheat plant. Symbols of stress such as proline, malondialdehyde (MDA) and antioxidant enzymes, which may be complicated in the suppression responses of plants, were also studied. Wheat plants were exposed to 0, 2500, 5000, 7500, 10,000 and 15,000 µM of Ce and Sm for 7 days. The growth enhanced in plants treated with lesser Ce and Sm concentration (2500 µM) and declined in plants treated with upper concentrations as compared to untreated plants. The treatment with 2500 µM of Ce and Sm increased dry weigh in Arta by 68.42 and 20% and in Baharan by 32.14% and 27.3%. Thus, Ce and Sm had hormesis effect on growth in wheat plants. According to plant's growth parameter patterns, Arta cultivar had more sensitive to Sm than to Ce, whereas Baharan cultivar had sensitive to Ce than to Sm. Our results indicated impact of Ce and Sm on proline accumulation depended on the dosage of Ce and Sm. It was observed that Ce and Sm accumulated in wheat plants at higher exposure doses. Increment of MDA content by Ce and Sm treatments showed that these metals caused oxidative stress in wheat plants. Ce and Sm blocked enzymatic antioxidant system (superoxide dismutases, peroxidase and polyphenol peroxidase) in wheat. In wheat plants treated with lower Ce and Sm concentrations higher amounts of non-enzymatic antioxidant metabolites were detected. Thus, we showed the potential negative impact of unsuitable utilization of REEs in plants and suggested growth and interruption in physiological and biochemical mechanisms as a possible factor to recognize the underlying toxicological processes.

Lactobacillus paracasei L9 affects disease progression in experimental autoimmune neuritis by regulating intestinal flora structure and arginine metabolism.

BACKGROUND: Autoimmune neuropathies are common peripheral nervous system (PNS) disorders. Environmental influences and dietary components are known to affect the course of autoimmune diseases. Intestinal microorganisms can be dynamically regulated through diet, and this study combines intestinal microorganisms with diseases to open up new therapeutic ideas. METHODS: In Lewis rats, a model of EAN was established with P0 peptide, Lactobacillus were used as treatment, serum T-cell ratio, inflammatory factors, sciatic neuropathological changes, and pathological inflammatory effects on intestinal mucosa were detected, and fecal metabolomics and 16 s microbiome analysis were performed to further explore the mechanism. RESULTS: In the EAN rat model, Lactobacillus paracasei L9 (LP) could dynamically regulate the CD4+/CD8+T balance in serum, reduce serum IL-1, IL-6 and TNF-α expression levels, improve sciatic nerve demyelination and inflammatory infiltration, and reduce nervous system score. In the rat model of EAN, intestinal mucosa was damaged. Occludin and ZO-1 were downregulated. IL-1, TNF-α and Reg3γ were upregulated. LP gavage induced intestinal mucosa recovery; occludin and ZO-1 upregulation; IL-1, TNF-α and Reg3γ downregulation. Finally, metabolomics and 16 s microbiome analysis were performed, and differential metabolites were enriched with an important metabolic pathway, arginine and proline metabolism. CONCLUSION: LP improved EAN in rats by influencing intestinal community and the lysine and proline metabolism.

Saline-Alkali Soil Property Improved by the Synergistic Effects of Priestia aryabhattai JL-5, Staphylococcus pseudoxylosus XW-4, Leymus chinensis and Soil Microbiota.

Two saline-alkali-tolerant bacterial strains, Priestia aryabhattai JL-5 and Staphylococcus pseudoxylosus XW-4, were isolated, with high capabilities of hydrolyzing phosphate and producing cellulase, respectively. The molecular mechanisms regulating the saline-alkali tolerance in the strain JL-5 were further investigated using transcriptome analysis. The contents of lactic acid and proline and the enzymatic activity of glutamine synthetase in the strain JL-5 were significantly increased. The properties of saline-alkali soils were significantly improved by the enhanced growth of the indicator plant Leymus chinensis under the combined applications of the strains JL-5 and XW-4 mixed with corn straw. The contents of catalase, peroxidase, superoxide dismutase and proline of L. chinensis were significantly increased, and the content of malondialdehyde was significantly decreased in the combined treatment of both bacterial strains. The contents of available nitrogen, phosphorus and potassium and organic matters in the soil treated with both strains were significantly increased, as well as the diversity and abundance of the soil microbiota. Our study evidently demonstrated the synergistic effects of the strains JL-5 and XW-4, indicator plants and the local microbiota in terms of improving the saline-alkali soil properties, providing strong experimental evidence to support the commercial development of the combined application of both strains to improve the properties of saline-alkali soils.

Influence of Deamidation on the Formation of Pyrazines and Proline-Specific Compounds in Maillard Reaction of Asparagine and Proline with Glucose.

Maillard reaction products obtained from the model system of binary amino acids (asparagine and proline) with glucose were first studied. GC-MS results showed that proline-specific aromatic compounds, 2,3-dihydro-1H-pyrrolizines and cyclopent[b]azepin-8(1H)-ones, were dominant among overall products, followed by pyrazines at different temperatures. Aspartic acid was first applied to model reactions as the precise control of asparagine deamidation, and lysine was further introduced into model systems for improving pyrazine formation. Quantitative results of model reaction products demonstrated that pyrazines were not significantly increased in deamidated states (Asn-Asp-Pro and Asp-Pro) while proline-specific compounds had a rapid enhancement at the same time. With excellent ability to form pyrazines, lysine did help to increase the formation of pyrazines, but still far fewer than pyrrolizines and azepines. It was assumed that proline would preferentially react with α-dicarbonyl compounds in Maillard reaction cascades with lower activation energies.

Evaluation of waterlogging tolerance and responses of protective enzymes to waterlogging stress in pumpkin.

Waterlogging caused by short and severe, or prolonged precipitation can be attributed to global warming. Pumpkin plants are drought-tolerant but not tolerate to waterlogging stress. Under frequent rain and waterlogging conditions, the production of pumpkins is of lower quality, sometimes rotten, and harvest failure occurs in severe cases. Therefore, it is of great significance to assess the waterlogging tolerance mechanism of pumpkin plants. In this study, 10 novel pumpkin varieties from Baimi series were used. The waterlogging tolerance level of pumpkin plants was evaluated by measuring waterlogging tolerance coefficient of biomass and physiological indices using waterlogging stress simulation method. The criteria to evaluate the waterlogging tolerance capacities of pumpkin plants were also explored. Using principal component and membership function analysis, waterlogging tolerance levels of the pumpkin varieties were ranked as follows: Baimi No. 10>; Baimi No. 5>; Baimi No. 1>; Baimi No. 2>; Baimi No. 3>; Baimi No. 7>; Baimi No. 9>; Baimi No. 6>; Baimi No. 4>; Baimi No. 8. Based on the results, Baimi No. 10 was identified with strong waterlogging tolerance and Baimi No. 8 with weak waterlogging tolerance. The responses of malondialdehyde (MDA), proline, key enzymes responsible for anaerobic respiration, and antioxidant enzymes to waterlogging stress were studied in pumpkin plants. The relative expression levels of related genes were determined using real-time fluorescence quantitative PCR technique. The aim of our study was to assess the waterlogging tolerance mechanism of pumpkin plants, thus laying a theoretical foundation for breeding waterlogging-tolerant varieties in the future. After flooding stress treatment, the antioxidant enzyme activities, contents of proline and alcohol dehydrogenases of Baimi No. 10 and Baimi No. 8 displayed an increase followed by a decrease. All indices of Baimi No. 10 were higher than Baimi No. 8. MDA contents gradually increased, with the content being higher in Baimi No. 8 than Baimi No. 10. The activities of pyruvate decarboxylases (PDCs) in Baimi No. 8 and Baimi No. 10 exhibited a decrease initially, followed by an increase, and then a decrease again. The PDC activity in Baimi No. 8 was generally higher than Baimi No. 10. The relative expression levels of genes encoding superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase were consistent with their corresponding enzyme activities. During the early stage of flooding stress, pumpkin plants waterlogging tolerance was improved by enhancing the expression levels of antioxidant enzyme encoding genes and increasing the antioxidant enzyme activities.

Substrate use and temperature effects in flight muscle mitochondria from an endothermic insect, the hawkmoth Manduca sexta.

Manduca sexta are endothermic insects, requiring adult thorax temperatures to be elevated above 35 °C for flight muscles to produce the wing beat frequencies necessary for flight. During flight, these animals rely on aerobic production of ATP by flight muscle mitochondria with several potential metabolic pathways providing the fuel. Along with typical carbohydrate substrates, mitochondria of other endothermic insects including bumblebees and wasps can use the amino acid proline or glycerol 3-phosphate (G3P) as metabolic fuel for prewarm up and flight. Here we examine flight muscle mitochondria physiology and the role of temperature and substrates in oxidative phosphorylation from 3-day old adult Manduca sexta. Mitochondria oxygen flux from flight muscle fibers were temperature sensitive with Q10 values ranging from 1.99 to 2.90, with a large increase in LEAK respiration with increased temperature. Mitochondria oxygen flux was stimulated by carbohydrate-based substrates, with flux through Complex I substrates providing the greatest oxygen flux. Neither proline nor G3P produced an increase in oxygen flux of the flight muscle mitochondria. Unlike other endothermic insects, Manduca are unable to supplement carbohydrate oxidation with either proline or G3P entering through Coenzyme Q and rely on substrates entering at complex I and II.

Enhanced Molecular Networking Shows Microbacterium sp. V1 as a Factory of Antioxidant Proline-Rich Peptides.

Two linear proline-rich peptides (1-2), bearing an N-terminal pyroglutamate, were isolated from the marine bacterium Microbacterium sp. V1, associated with the marine sponge Petrosia ficiformis, collected in the volcanic CO2 vents in Ischia Island (South Italy). Peptide production was triggered at low temperature following the one strain many compounds (OSMAC) method. Both peptides were detected together with other peptides (3-8) via an integrated, untargeted MS/MS-based molecular networking and cheminformatic approach. The planar structure of the peptides was determined by extensive 1D and 2D NMR and HR-MS analysis, and the stereochemistry of the aminoacyl residues was inferred by Marfey's analysis. Peptides 1-8 are likely to arise from Microbacterium V1 tailor-made proteolysis of tryptone. Peptides 1 and 2 were shown to display antioxidant properties in the ferric-reducing antioxidant power (FRAP) assay.

Collagen-Derived Dipeptides and Amino Acids Have Immunomodulatory Effects in M1-Differentiated RAW264.7 Cells and PBMC.

A number of food components, such as polyphenols and phytonutrients, have immunomodulatory effects. Collagen has various bioactivities, such as antioxidative effects, the promotion of wound healing, and relieving symptoms of bone/joint disease. Collagen is digested into dipeptides and amino acids in the gastrointestinal tract and subsequently absorbed. However, the difference in immunomodulatory effects between collagen-derived dipeptides and amino acids is unknown. To investigate such differences, we incubated M1 macrophages or peripheral blood mononuclear cells (PBMC) with collagen-derived dipeptides (hydroxyproline-glycine (Hyp-Gly) and proline-hydroxyproline (Pro-Hyp)) and amino acids (proline (Pro), hydroxyproline (Hyp), and glycine (Gly)). We first investigated the dose dependency of Hyp-Gly on cytokine secretion. Hyp-Gly modulates cytokine secretion from M1 macrophages at 100 µM, but not at 10 µM and 1 µM. We then compared immunomodulatory effects between dipeptides and mixtures of amino acids on M1 macrophages and PBMC. There was, however, no difference in cytokine secretion between dipeptides and their respective amino acids. We conclude that collagen-derived dipeptides and amino acids have immunomodulatory effects on M1-differentiated RAW264.7 cells and PBMC and that there is no difference in the immunomodulatory effects between dipeptides and amino acids.

1H NMR-based urinary metabolic analysis of high-dose cyclophosphamide-induced toxicity in mice.

Cyclophosphamide (CP) is widely used in clinical fields. Beside its therapeutic effects, CP shows toxicity depending on dose and administration schedule. In this study, the urinary metabolic profiles were investigated in mice intraperitoneally injected with high-dose CP (150 mg/kg body weight) once a week over four weeks using nuclear magnetic resonance (NMR)-based metabolomics. Twenty-six metabolites were identified as potential biomarkers by multivariate statistical analysis. A decrease in isoleucine, alanine, N-acetylglutamic acid, proline, methionine, valine, phenylacetylglulamine, dimethylamine, hippurate, acetic acid, lactate, α-oxoglutarate, citrate, malonic acid, creatinine, niacin, ß-hydroxybutyrate, and betaine, whereas an increase in leucine, glutamate, glycine, taurine, phenylacetylglycine, glucose, creatine, and choline were observed in the urine of high-dose CP-treated mice. Metabolites related to amino acid metabolism, energy metabolism, and gut microbial metabolism were changed markedly in the urine. Further metabolic pathway analysis suggested that seven metabolic pathways, including alanine, aspartate, and glutamate metabolism, arginine biosynthesis, glyoxylate, and dicarboxylate metabolism, glycine, serine and threonine metabolism, d-glutamine and d-glutamate metabolism, arginine, and proline metabolism, citrate cycle, as well as the gut microbiota metabolism, were significantly involved in response to high-dose CP treatment. These findings help to predict the toxicity of CP and understand the biological mechanism of the toxicity of CP.

Enantioseparation of a-substituted proline analogs with macrocyclic glycopeptide-based chiral stationary phases immobilized on superficially porous particles of silica applying liquid chromatography with ultraviolet and mass spectrometric detection.

In this study, the liquid chromatography-based direct enantioseparation of the stereoisomers of α-substituted proline analogs has been investigated utilizing chiral stationary phases with UV and/or mass spectrometric (MS) detection. Macrocyclic antibiotics, such as vancomycin, teicoplanin, modified teicoplanin, and teicoplanin aglycone, all covalently immobilized to 2.7 µm superficially porous silica particles have been applied as stationary phases. Mobile phases utilizing mixtures of methanol and acetonitrile with different additives (polar-ionic mode) were optimized during method development. Best separations were achieved with mobile phases of 100% MeOH containing either 20 mM acetic acid or 20 mM triethylammonium acetate. Special attention was given to the applicability of MS-compatible mobile phases. Acetic acid was found to be advantageous as a mobile phase additive for MS detection. Enantioselective chromatographic behaviors are interpreted based on the explored correlations between the analytes' structural features and those of the applied chiral stationary phases. For the thermodynamic characterization, separations were studied in the temperature range of 5-50 °C. Generally, retention and selectivity decreased with increasing temperature, and in most cases, enthalpy-driven enantiorecognition was observed, but entropic contributions also were present. Unexpectedly, unusual shapes for the van Deemter curves were registered in the kinetic evaluations. General trends could be observed in the enantiomeric elution orders: S < R on VancoShell and NicoShell, and opposite R < S on TeicoShell and TagShell columns.

Overexpression of NtGCN2 improves drought tolerance in tobacco by regulating proline accumulation, ROS scavenging ability, and stomatal closure.

Drought stress is a severe threat to plants. Genes that respond to drought stress are essential for plant growth and development. General control nonderepressible 2 (GCN2) encodes a protein kinase that responds to various biotic and abiotic stresses. However, the mechanism of GCN2 in plant drought tolerance remains unclear. In the present study, the promoters of NtGCN2 from Nicotiana tabacum K326, which contained a drought-responsive Cis-acting element MYB that can be activated by drought stress, were cloned. Furthermore, the drought tolerance function of NtGCN2 was investigated using NtGCN2-overexpressed transgenic tobacco plants. NtGCN2-overexpressed transgenic plants were more tolerant to drought stress than wild-type (WT) plants. The transgenic tobacco plants exhibited higher proline and abscisic acid (ABA) contents, antioxidant enzyme activities, leaf relative water content, and expression levels of genes encoding key antioxidant enzymes and proline synthase, but lower levels of malondialdehyde and reactive oxygen species, and reduced stomatal apertures, stomatal densities, and stomatal opening rates compared to WT plants under drought stress. These results indicated that overexpression of NtGCN2 conferred drought tolerance in transgenic tobacco plants. RNA-seq analysis showed that overexpression of NtGCN2 responded to drought stress by regulating the expression of genes related to proline synthesis and catabolism, abscisic acid synthesis and catabolism, antioxidant enzymes, and ion channels in guard cells. These results showed that NtGCN2 might regulate drought tolerance by regulating proline accumulation, reactive oxygen species (ROS) scavenging, and stomatal closure in tobacco and may have the potential for application in the genetic modification of crop drought tolerance.