RESUMEN
Escherichia coli uses allantoin as the sole nitrogen source during anaerobic growth. In the final step of allantoin degradation, oxamic transcarbamylase (OXTCase) converts oxalurate to carbamoyl phosphate (CP) and oxamate. The activity of this enzyme was first measured in Streptococcus allantoicus in the 1960s, but no OXTCase enzyme or the encoding gene(s) have been found in any strain. This study discovered that allFGH (fdrA, ylbE, and ylbF) are the genes that encode the global orphan enzyme OXTCase. The three genes form an operon together with allK (ybcF), encoding catabolic carbamate kinase. The allFGHK operon is located directly downstream of the allECD operon that encodes enzymes for the preceding steps of OXTCase. The OXTCase kinetic parameters were analyzed using the purified protein composed of AllF-AllG-AllH (FdrA-YlbE-YlbF); for the substrate CP, KM and Vmax were 1.3 mM and 15.4 U/mg OXTCase, respectively, and for the substrate oxamate, they were 36.9 mM and 27.0 U/mg OXTCase. In addition, the OXTCase encoded by the three genes is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase, ornithine transcarbamylase, and YgeW transcarbamylase. The present study elucidated the anaerobic allantoin degradation pathway of E. coli. Therefore, we suggest that the genes fdrA, ylbE, and ylbF are renamed allF, allG, and allH, respectively.IMPORTANCEThe anaerobic allantoin degradation pathway of Escherichia coli includes a global orphan enzyme, oxamic transcarbamylase (OXTCase), which converts oxalurate to carbamoyl phosphate and oxamate. This study found that the allFGH (fdrA, ylbE, and ylbF) genes encode OXTCase. The OXTCase activity and kinetics were successfully determined with purified recombinant AllF-AllG-AllH (FdrA-YlbE-YlbF). This OXTCase is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase (ATCase), ornithine transcarbamylase (OTCase), and YgeW transcarbamylase (YTCase). In addition, OXTCase activity requires three genes, whereas ATCase is encoded by two genes, and OTCase and YTCase are encoded by a single gene. The current study discovered OXTCase, the last unknown step in allantoin degradation, and this enzyme is a new member of the transcarbamylase group that was previously unknown.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Alantoína/metabolismo , Operón , CinéticaRESUMEN
Ureides, the degraded products of purine catabolism in Arabidopsis, have been shown to act as antioxidant and nitrogen sources. Herein we elucidate purine degraded metabolites as a carbon source using the Arabidopsis Atxdh1, Ataln, and Ataah knockout (KO) mutants vis-à-vis wild-type (WT) plants. Plants were grown under short-day conditions on agar plates containing half-strength MS medium with or without 1% sucrose. Notably, the absence of sucrose led to diminished biomass accumulation in both shoot and root tissues of the Atxdh1, Ataln, and Ataah mutants, while no such effect was observed in WT plants. Moreover, the application of sucrose resulted in a reduction of purine degradation metabolite levels, specifically xanthine and allantoin, predominantly within the roots of WT plants. Remarkably, an increase in proteins associated with the purine degradation pathway was observed in WT plants in the presence of sucrose. Lower glyoxylate levels in the roots but not in the shoot of the Atxdh1 mutant in comparison to WT, were observed under sucrose limitation, and improved by sucrose application in root, indicating that purine degradation provided glyoxylate in the root. Furthermore, the deficit of purine-degraded metabolites in the roots of mutants subjected to carbon starvation was partially mitigated through allantoin application. Collectively, these findings signify that under conditions of sucrose limitation and short-day growth, purines are primarily remobilized within the root system to augment the availability of ureides, serving as an additional carbon (as well as nitrogen) source to support plant growth.
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Arabidopsis , Carbono , Raíces de Plantas , Sacarosa , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Carbono/metabolismo , Sacarosa/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Alantoína/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Purinas/metabolismo , Urea/metabolismo , Brotes de la Planta/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Glioxilatos/metabolismoRESUMEN
BACKGROUND: Dioscorea opposita Thunb. cv. Tiegun maturity (DM) is an important factor influencing its quality. However, there are few studies on the impact of harvest time on its maturation. In the present study, a NMR-based metabolomics approach was applied to investigate the dynamic metabolic changes of D. opposita Thunb. cv. Tiegun at six different harvest stages: stage 1 (S1), stage 2 (S2), Stage 3 (S3), stage 4 (S4), stage 5 (S5) and stage 6 (S6). RESULTS: Principal component analysis showed distinct segregation of samples obtained from S1, S2 and S3 compared to those derived from S4, S5 and S6. Interestingly, these samples from the two periods were obtained before and after frost, indicating that frost descent might be important for DM. Eight differential metabolites responsible for good separation of different groups were identified by the principal component analysis loading plot and partial least squares-discriminant analysis. In addition, quantitative analysis of these metabolites using liquid chromatography-tandem mass spectrometry determined the effects of harvest time on these metabolite contents, two of which, sucrose and allantoin, were considered as potential biomarkers to determine DM. CONCLUSION: The present study demonstrated that NMR-based metabolomics approach could serve as a powerful tool to identify differential metabolites during harvesting processes, also offering a fresh insight into understanding the DM and the potential mechanism of quality formation. © 2024 Society of Chemical Industry.
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Dioscorea , Espectroscopía de Resonancia Magnética , Metabolómica , Espectrometría de Masas en Tándem , Dioscorea/química , Dioscorea/metabolismo , Dioscorea/crecimiento & desarrollo , Espectroscopía de Resonancia Magnética/métodos , Frutas/química , Frutas/metabolismo , Frutas/crecimiento & desarrollo , Alantoína/metabolismo , Alantoína/análisis , Factores de Tiempo , Sacarosa/metabolismo , Sacarosa/análisis , Cromatografía Liquida/métodos , Análisis de Componente Principal , Cromatografía Líquida de Alta Presión , Cromatografía Líquida con Espectrometría de MasasRESUMEN
This study investigated the impact of various enhancers on permeation through the skin and accumulation in the skin from acrylic pressure-sensitive adhesive-based drug-in-adhesives matrix-type transdermal patches. Eleven patches, each containing a 5% enhancer of permeation, encompassing compounds such as salicylic acid, menthol, urea, glycolic acid, allantoin, oleic acid, Tween 80, linolenic acid, camphor, N-dodecylcaprolactam, and glycerin, were developed. Ibuprofen (IBU) was the model active substance, a widely-used non-steroidal anti-inflammatory drug. The results were compared to patches without enhancers and commercial preparations. The study aimed to assess the effect of enhancers on IBU permeability. The adhesive properties of the patches were characterised, and active substance permeability was tested. The findings revealed that patches with 5% allantoin exhibited the highest IBU permeability, approximately 2.8 times greater than patches without enhancers after 24 h. These patches present a potential alternative to commercial preparations, highlighting the significant impact of enhancers on transdermal drug delivery efficiency.
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Alantoína , Ibuprofeno , Ibuprofeno/farmacología , Alantoína/metabolismo , Administración Cutánea , Piel/metabolismo , Absorción Cutánea , Adhesivos/metabolismoRESUMEN
The ureides allantoin and allantoate serve as nitrogen (N) transport compounds in plants, and more recently, allantoin has been shown to play a role in signaling. In planta, tissue ureide levels are controlled by the activity of enzymes of the purine degradation pathway and by ureide transporters called ureide permeases (UPS). Little is known about the physiological function of UPS proteins in crop plants, and especially in monocotyledon species. Here, we identified 13 TaUPS genes in the wheat (Triticum aestivum L.) genome. Phylogenetic and genome location analyses revealed a close relationship of wheat UPSs to orthologues in other grasses and a division into TaUPS1, TaUPS2.1, and TaUPS2.2 groups, each consisting of three homeologs, with a total of four tandem duplications. Expression, localization, and biochemical analyses resolved spatio-temporal expression patterns of TaUPS genes, transporter localization at the plasma membrane, and a role for TaUPS2.1 proteins in cellular import of ureides and phloem and seed loading. In addition, positive correlations between TaUPS1 and TaUPS2.1 transcripts and ureide levels were found. Together the data support that TaUPSs function in regulating ureide pools at source and sink, along with source-to-sink transport. Moreover, comparative studies between wheat cultivars grown at low and high N strengthened a role for TaUPS1 and TaUPS2.1 transporters in efficient N use and in controlling primary metabolism. Co-expression, protein-protein interaction, and haplotype analyses further support TaUPS involvement in N partitioning, N use efficiency, and domestication. Overall, this work provides a new understanding on UPS transporters in grasses as well as insights for breeding resilient wheat varieties with improved N use efficiency.
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Alantoína , Proteínas de Transporte de Membrana , Alantoína/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Triticum/genética , Triticum/metabolismo , Nitrógeno/metabolismo , Filogenia , FitomejoramientoRESUMEN
Soil salinity stress is one of the major bottlenecks for crop production. Although, allantoin is known to be involved in nitrogen metabolism in plants, yet several reports in recent time indicate its involvement in various abiotic stress responses including salinity stress. However, the detail mechanism of allantoin involvement in salinity stress tolerance in plants is not studied well. Moreover, we demonstrated the role of exogenous application of allantoin as well as increased concentration of endogenous allantoin in rendering salinity tolerance in rice and Arabidopsis respectively, via., induction of abscisic acid (ABA) and brassinosteroid (BR) biosynthesis pathways. Exogenous application of allantoin (10 µM) provides salt-tolerance to salt-sensitive rice genotype (IR-29). Transcriptomic data after exogenous supplementation of allantoin under salinity stress showed induction of ABA (OsNCED1) and BR (Oscytochrome P450) biosynthesis genes in IR-29. Further, the key gene of allantoin biosynthesis pathway i.e., urate oxidase of the halophytic species Oryza coarctata was also found to induce ABA and BR biosynthesis genes when over-expressed in transgenic Arabidopsis. Thus, indicating that ABA and BR biosynthesis pathways were involved in allantoin mediated salinity tolerance in both rice and Arabidopsis. Additionally, it has been found that several physio-chemical parameters such as biomass, Na+/K+ ratio, MDA, soluble sugar, proline, allantoin and chlorophyll contents were also associated with the allantoin-mediated salinity tolerance in urate oxidase overexpressed lines of Arabidopsis. These findings depicted the functional conservation of allantoin for salinity tolerance in both plant clades.
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Arabidopsis , Oryza , Arabidopsis/metabolismo , Ácido Abscísico/farmacología , Ácido Abscísico/metabolismo , Oryza/genética , Oryza/metabolismo , Tolerancia a la Sal/genética , Alantoína/metabolismo , Brasinoesteroides/farmacología , Brasinoesteroides/metabolismo , Urato Oxidasa/genética , Urato Oxidasa/metabolismo , Proteínas de Plantas/metabolismo , Estrés Fisiológico/genética , Salinidad , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Fracture healing is a rigorous and orderly process with multiple steps that are mediated by multiple cells. During this process, osteoclast-mediated bone remodeling plays a critical role, and its abnormal activity leads not only to fracture susceptibility but also to impaired fracture healing. However, few studies have focused on impaired healing caused by osteoclast defects, and clinical drugs for this type of impaired fracture healing are still lacking. The cell types and regulatory pathways in the zebrafish skeletal system are highly similar to those of mammals, making the zebrafish skeletal system being widely used for skeletal-related studies. To study the process of fracture healing disorders caused by osteoclast defects and discover potential therapeutic drugs, we established an in vivo osteoclast-deficient fracture model using a previously generated fms gene mutant zebrafish (fmsj4e1). The results showed that reduced functional osteoclasts could affect fracture repair in the early stages of fracture. Then we applied an in vitro scale culture system to screen for osteoclast-activating drugs. We found the small molecule compound allantoin (ALL) being able to activate osteoclasts. Subsequently, we verified the activation role of ALL on osteoclasts and the promotion of fracture repair in an in vivo fmsj4e1 fracture defect model. Finally, by examining the osteoclastogenesis and maturation process, we found that ALL may promote osteoclast maturation by regulating RANKL/OPG, thus promoting fmsj4e1 fracture healing. Our study provides a potential new approach for the future improvement of fracture healing disorders caused by osteoclast defects.
Asunto(s)
Osteoclastos , Pez Cebra , Animales , Osteoclastos/metabolismo , Curación de Fractura , Alantoína/metabolismo , Osteogénesis , Diferenciación Celular/genética , MamíferosRESUMEN
Purines are abundant among organic nitrogen sources and have high nitrogen content. Accordingly, microorganisms have evolved different pathways to catabolize purines and their metabolic products such as allantoin. Enterobacteria from the genera Escherichia, Klebsiella and Salmonella have three such pathways. First, the HPX pathway, found in the genus Klebsiella and very close relatives, catabolizes purines during aerobic growth, extracting all four nitrogen atoms in the process. This pathway includes several known or predicted enzymes not previously observed in other purine catabolic pathways. Second, the ALL pathway, found in strains from all three species, catabolizes allantoin during anaerobic growth in a branched pathway that also includes glyoxylate assimilation. This allantoin fermentation pathway originally was characterized in a gram-positive bacterium, and therefore is widespread. Third, the XDH pathway, found in strains from Escherichia and Klebsiella spp., at present is ill-defined but likely includes enzymes to catabolize purines during anaerobic growth. Critically, this pathway may include an enzyme system for anaerobic urate catabolism, a phenomenon not previously described. Documenting such a pathway would overturn the long-held assumption that urate catabolism requires oxygen. Overall, this broad capability for purine catabolism during either aerobic or anaerobic growth suggests that purines and their metabolites contribute to enterobacterial fitness in a variety of environments.
Asunto(s)
Alantoína , Ácido Úrico , Alantoína/metabolismo , Ácido Úrico/metabolismo , Enterobacteriaceae/metabolismo , Purinas/metabolismo , Nitrógeno/metabolismoRESUMEN
Drought is a misfortune for agriculture and human beings. The annual crop yield reduction caused by drought exceeds the sum of all pathogens. As one of the gatekeepers of China's "granary", rice is the most important to reveal the key drought tolerance factors in rice. Rice seedlings of Nipponbare (Oryza sativa L. ssp. Japonica) were subjected to simulated drought stress, and their root systems were analyzed for the non-targeted metabolome and strand-specific transcriptome. We found that both DEGs and metabolites were enriched in purine metabolism, and allantoin accumulated significantly in roots under drought stress. However, few studies on drought tolerance of exogenous allantoin in rice have been reported. We aimed to further determine whether allantoin can improve the drought tolerance of rice. Under the treatment of exogenous allantoin at different concentrations, the drought resistant metabolites of plants accumulated significantly, including proline and soluble sugar, and reactive oxygen species (ROS) decreased and reached a significant level in 100 µmol L-1. To this end, a follow-up study was identified in 100 µmol L-1 exogenous allantoin and found that exogenous allantoin improved the drought resistance of rice. At the gene level, under allantoin drought treatment, we found that genes of scavenge reactive oxygen species were significantly expressed, including peroxidase (POD), catalase (CATA), ascorbate peroxidase 8 (APX8) and respiratory burst oxidase homolog protein F (RbohF). This indicates that plants treated by allantoin have better ability to scavenge reactive oxygen species to resist drought. Alternative splicing analysis revealed a total of 427 differentially expressed alternative splicing events across 320 genes. The analysis of splicing factors showed that gene alternative splicing could be divided into many different subgroups and play a regulatory role in many aspects. Through further analysis, we restated the key genes and enzymes in the allantoin synthesis and catabolism pathway, and found that the expression of synthetase and hydrolase showed a downward trend. The pathway of uric acid to allantoin is completed by uric acid oxidase (UOX). To find out the key transcription factors that regulate the expression of this gene, we identified two highly related transcription factors OsERF059 and ONAC007 through correlation analysis. They may be the key for allantoin to enhance the drought resistance of rice.
Asunto(s)
Alantoína , Oryza , Estrés Fisiológico , Humanos , Alantoína/metabolismo , Alantoína/farmacología , Estudios de Seguimiento , Regulación de la Expresión Génica de las Plantas , Oryza/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Transcriptoma , Ácido Úrico/metabolismo , Metaboloma , Fenómenos Fisiológicos de las Plantas/genéticaRESUMEN
The present study was intended to evaluate the effect of forage source (alfalfa hay; ALF vs. corn silage; CS) along with a supplemental fat source (soybean oil; SO vs. rumen-inert palm fatty acids; PF) on growth performance, nutrient digestibility, and ruminal fermentation in dairy calves. Forty-eight new-born Holstein female calves (3 d old) were assigned to one of 4 treatments: (1) alfalfa hay with soybean oil (ALF-SO); (2) alfalfa hay with palm fatty acids (ALF-PF); (3) corn silage with soybean oil (CS-SO); (4) corn silage with palm fatty acids (CS-PF). Starter diets had equal amounts of forage (100 g/kg dry matter; DM) and fat source (30 g/kg DM). Calves were fed a constant amount of milk (d 1 to 63) and had ad libitum access to water and starters (d 1 to 83). The lowest and greatest starter intakes during the preweaning period occurred in ALF-SO and CS-PF, respectively. This coincided with forage × fat source interaction for average daily gain (ADG) during preweaning. The forage source affected total DM intake and ADG over the entire period, body weight (BW) at weaning, and final BW with greater values in calves that received CS compared with ALF. The concentrations of total short-chain fatty acids and butyrate were increased, whereas concentration of acetate and acetate:propionate ratio were decreased in the rumen of calves fed CS compared with ALF. Feeding CS increased urinary excretion of allantoin and, as a trend, total purine derivatives (PD) and estimated microbial protein synthesis in comparison with ALF. The fat source affected starter intake, ADG, and BW postweaning with the highest values in PF. The digestibility of neutral detergent fiber, crude protein and, as a trend, organic matter were higher in calves fed PF compared with SO. Calves fed PF had lower ruminal ammonia-N concentration and urinary N excretion and greater urinary excretion of allantoin and total PD. Calves receiving SO had a lower ruminal protozoa population. In conclusion, supplementing starter diets with CS and PF is superior to ALF and SO. Interaction of the positive effects of CS and PF on performance underlines that concurrent supplementation of CS with PF is especially recommendable in young calves before weaning.
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Ensilaje , Zea mays , Bovinos , Animales , Femenino , Ensilaje/análisis , Zea mays/metabolismo , Fermentación , Medicago sativa/metabolismo , Rumen/metabolismo , Aceite de Soja/metabolismo , Ácidos Grasos/metabolismo , Alimentación Animal/análisis , Alantoína/metabolismo , Dieta/veterinaria , Nutrientes , Peso CorporalRESUMEN
Tropical legumes transport fixed nitrogen in form of ureides (allantoin and allantoate) over long distances from the nodules to the shoot. Ureides are formed in nodules from purine mononucleotides by a partially unknown reaction network that involves bacteroid-infected and uninfected cells. Here, we demonstrate by metabolic analysis of CRISPR mutant nodules of Phaseolus vulgaris defective in either xanthosine monophosphate phosphatase (XMPP), guanosine deaminase (GSDA), the nucleoside hydrolases 1 and 2 (NSH1, NSH2) or xanthine dehydrogenase (XDH) that nodule ureide biosynthesis involves these enzymes and requires xanthosine and guanosine but not inosine monophosphate catabolism. Interestingly, promoter reporter analyses revealed that XMPP, GSDA and XDH are expressed in infected cells, whereas NSH1, NSH2 and the promoters of the downstream enzymes urate oxidase (UOX) and allantoinase (ALN) are active in uninfected cells. The data suggest a complex cellular organization of ureide biosynthesis with three transitions between infected and uninfected cells.
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Nitrógeno , Phaseolus , Alantoína/metabolismo , N-Glicosil Hidrolasas/metabolismo , Nitrógeno/metabolismo , Phaseolus/genética , Xantina Deshidrogenasa/metabolismoRESUMEN
Alzheimer's disease (AD) is a brain disease that causes problems in memory, thinking, and behavior. Allantoin has been shown to have antioxidant, anti-inflammatory, and neuroprotective effects. In this study, we aimed to investigate the effect and mechanism of action of allantoin on AD-related memory impairment. We investigated the effect of allantoin on an amyloid ß1-42 peptide (Aß1-42)-induced AD model in rats and evaluated its memory-enhancing effect using the Morris water maze test. Pathological changes in the hippocampus and cortex were examined by hematoxylin-eosin staining. The expression of the phosphorylated Tau protein and PI3K/Akt/GSK-3ß signaling pathway was analyzed by western blotting. The results of the water maze test showed that after treatment with allantoin, the rats could reduce their swimming time and travel distances to find the platform. Allantoin treatment also increased the time spent in the quadrant in which the platform was located. Histological assessment showed that Aß1-42 could cause morphological alterations in nerve cells in the hippocampal CA1 region, and that allantoin could repair the damage to these cells. Western blotting revealed that allantoin treatment increased the expression of p-PI3K, p-Akt, and p-GSK-3ß and decreased p-Tau in the hippocampus and cortex of rats. These effects were inhibited by LY294002. These findings showed that allantoin could improve cognitive impairment in Aß1-42-induced rats by activating the PI3K/Akt/GSK-3ß signaling pathway to reduce abnormal hyperphosphorylation of Tau. Thus, allantoin may be a potential therapeutic agent for neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Alantoína/metabolismo , Alantoína/farmacología , Alantoína/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hipocampo , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal , Proteínas tau/metabolismoRESUMEN
Pseudoallergic reactions are hypersensitivity reactions mediated by an IgE-independent mechanism. Since allantoin (AT)-mediated pseudoallergy has not been studied, in this study, our objective is to investigate the anti-pseudoallergy effect of AT and its underlying mechanism. In vitro, ß-hexosaminidase (ß-Hex) and histamine (HIS) release assays, inflammatory cytokine assays, toluidine blue staining, and F-actin microfilament staining were used to evaluate the inhibitory effect of AT in RBL-2H3 cells stimulated with Compound 48/80 (C48/80). Western blot analysis is further performed to investigate intracellular calcium fluctuation-related signaling pathways. In vivo, Evans Blue extraction, paw swelling, and the diameter of Evans Blue extravasation were evaluated, and skin tissues are examined for histopathological examination in mice with passive cutaneous anaphylaxis (PCA) induced by C48/80. Body temperature is measured, and the levels of cytokines are further determined by ELISA kits in mice with active systemic anaphylaxis (ASA) induced by C48/80. The results show that AT dose-dependently inhibited degranulation in C48/80-stimulated RBL-2H3 cells by inhibiting ß-Hex and HIS release, reducing the levels of TNF-α, IL-8, and MCP-1, inhibiting shape changes due to degranulation and disassembling the F-actin cytoskeleton. Furthermore, AT dose-dependently inhibits the phosphorylation of PLCγ and IP3R. In vivo, AT decreased Evans Blue extravasation, paw swelling, and the diameter of Evans Blue extravasation and significantly ameliorate pathological changes and mast cell degranulation in C48/80-induced PCA. Furthermore, AT help the mice recover from the C48/80-induced decrease in body temperature and decreased the levels of cytokines in C48/80-treated ASA mice. Our results indicate that allantoin inhibits compound 48/80-induced pseudoallergic reactions. AT has the potential to be used in IgE-independent anti-allergic and anti-inflammatory therapies.
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Anafilaxia , p-Metoxi-N-metilfenetilamina , Alantoína/metabolismo , Anafilaxia/inducido químicamente , Anafilaxia/tratamiento farmacológico , Anafilaxia/metabolismo , Animales , Degranulación de la Célula , Citocinas/metabolismo , Edema/patología , Azul de Evans/efectos adversos , Azul de Evans/metabolismo , Inmunoglobulina E/metabolismo , Mastocitos , Ratones , beta-N-Acetilhexosaminidasas/metabolismo , p-Metoxi-N-metilfenetilamina/efectos adversosRESUMEN
The aln-3 mutant overaccumulating allantoin and respective wild type (WT) strain of Arabidopsis thaliana were exposed to cadmium (Cd) or mercury (Hg) with or without nitric oxide (NO) donor (sodium nitroprusside, SNP) to study cross-talk, metabolic and oxidative changes between these nitrogen sources (organic vs. inorganic). The aln-3 accumulated over 10-fold more allantoin than WT with the effect of Cd and Hg differing in leaf and root tissue: aln-3 contained more ascorbic acid and phytochelatins when treated with Cd or Hg and more Cd in both organs. SNP depleted leaf Cd and root Hg accumulation in aln3 but had a positive impact on the amount of metabolites typically in WT plants, indicating potentially negative relation between allantoin and NO. In agreement, aln-3 roots showed lower NO signals in control or metal treatments, but higher ROS signal, and SNP had more pronounced impact in WT roots. Flavonol glycosides were more abundant in aln-3 and were affected more by metals than by SNP. Malate was the most affected Krebs acid with strong reaction to SNP and Hg treatment. Data indicate that allantoin overaccumulation influences the accumulation of specific metabolites but nitric oxide has a greater impact on the metabolite profile in WT.
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Arabidopsis , Mercurio , Alantoína/metabolismo , Alantoína/farmacología , Arabidopsis/metabolismo , Cadmio/metabolismo , Mercurio/metabolismo , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Raíces de Plantas/metabolismoRESUMEN
Allantoin is an important fine chemical that can be widely used in pharmaceutical, cosmetic and agricultural industries. Currently, allantoin is mainly produced by plant extraction or chemical synthesis. Due to the cost and environmental concerns, biosynthesis of allantoin from renewable feedstock is much more desirable. However, microbial production of allantoin from simple carbon sources has not yet been achieved so far. In this study, de novo biosynthesis of allantoin was achieved by constructing an artificial biosynthetic pathway. First, screening of efficient urate oxidases and xanthine dehydrogenases enabled allantoin production from hypoxanthine, a natural intermediate in purine metabolic pathway in Escherichia coli. Then, assemble of the entire pathway resulted in 13.9 mg/L allantoin from glucose in shake flask experiments. The titer was further improved to 639.8 mg/L by enhancing the supply of the precursor, redistribution of carbon flux, and reduction of acetate. Finally, scale-up production of allantoin was conducted in a 1-L fermentor under fed-batch culture conditions, which enabled the synthesis of 2360 mg/L allantoin, representing a 170-fold increase compared with the initial strain. This study not only demonstrates the potential for industrial production of allantoin, but also provides a bacterial platform for synthesis of other purines-derived high-value chemicals.
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Alantoína , Escherichia coli , Alantoína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas , Urato Oxidasa/genética , Urato Oxidasa/metabolismoRESUMEN
Legumes develop a symbiotic relationship with bacteria that are housed in root nodules and fix atmospheric di-nitrogen (N2) to ammonia. In soybean (Glycine max (L.) Merr.) nodules, the final products of nitrogen (N) fixation are amino acids, and the ureides allantoin and allantoic acid that also serve as the major long-distance N transport forms. Recently, we have shown that increased expression of UPS1 (ureide permease 1) in soybean nodules results in enhanced ureide export from nodules with positive effects on N fixation and seed yield. Here, we demonstrate that changes in the ureide transport processes trigger alterations in allantoin and allantoic acid pools and partitioning throughout the transgenic plants. They further result in adjustments in amino acid availability in, and translocation to, root and shoot sinks. In addition, leaf carbon (C) capture, assimilation and allocation to sinks are improved, accommodating the increased nodule function, and root and shoot growth. Overall, we demonstrate that enhanced ureide partitioning in nodulated soybean leads to a complex rebalancing of N and C acquisition, metabolism, and transport processes with positive consequences for above- and below-ground vegetative biomass, and whole-plant N and C gains.
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Alantoína/metabolismo , Glycine max/metabolismo , Nodulación de la Raíz de la Planta , Urea/análogos & derivados , Transporte Biológico , Nitrógeno/química , Nitrógeno/metabolismo , Fijación del Nitrógeno , Glycine max/microbiología , Urea/metabolismoRESUMEN
Purine degradation products have been shown to play roles in plant response to stresses such as drought, salinity, extended dark, nitrogen deficiency, and pathogen infection. In this study, we used Arabidopsis wild-type (WT) and an Atxdh1-knockout mutant defective in xanthine dehydrogenase1 (XDH1) to examine the role of degraded purine metabolites in the responses to wounding or UV-C stress applied to the middle leaves of the plant. Wounding or UV-C stress in the mutant resulted in lower fresh-weight, increased senescence symptoms, and increased cell death compared to WT plants. In addition, WT plants exhibited lower levels of oxidative stress indicators, reactive oxygen species, and malondialdehyde in their leaves than the mutant. Notably, transcripts and proteins functioning in the purine degradation pathway were regulated in such a way that it led to enhanced ureide levels in WT leaves 24h after applying the UV-C or wound stress. However, different remobilization of the accumulated ureides was observed after 72h of stress. In plants treated with UV-C, the concentration of allantoin was highest in young leaves, whereas in wounded plants it was lowest in these leaves and instead accumulated mainly in the middle leaves that had been wounded. These results indicated that in WT plants treated with UV-C, ureides were remobilized from the lower older and damaged leaves to support young leaf growth during the recovery period from stress. After wounding, however, whilst some ureides were remobilized to the young leaves, more remained in the wounded middle leaves to function as antioxidants and/or healing agents.
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Proteínas de Arabidopsis , Arabidopsis , Purinas/metabolismo , Rayos Ultravioleta/efectos adversos , Alantoína/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequías , Hojas de la Planta/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Wild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrAD296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrAD296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production. IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrAD296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the "orphan enzyme" oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.
Asunto(s)
Alantoína/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Ácido Succínico/metabolismo , Técnicas de Cocultivo , Escherichia coli/genética , Fermentación , Glicerol/metabolismo , Glioxilatos/metabolismo , Malatos/metabolismoRESUMEN
AIM: Following peripheral nerve injury, in addition to axonal and myelin degeneration, a sharp increase is observed in cell numbers, especially Schwann cells, in the distal part of the injury. This study investigated the effect of allantoin, involved in purine catabolism, on the reactions occurring in the lesion area. MATERIAL AND METHOD: An experimental sciatic nerve injury model was established with the application of pressure at 50 Newtons for 5 s to the right sciatic nerves of experimental animals following visualization with the help of pliers. Allantoin was administered to the test groups via the intraperitoneal (i.p.) route (10 mg/kg), at the same time every day for 30 days. The animals were sacrificed at the end of 30 days, following electromyography and Sciatic Function Index tests. Myelinated/unmyelinated axon numbers were evaluated stereologically. Myelin sheath thickness, axon diameter, mitotic activity, and functional improvement in muscles in this peripheral nerve degeneration model were investigated. The test results were then subjected to statistical analysis. RESULTS: Allantoin was observed to exhibit curative effects in terms of function, although stereological tests revealed no morphological differences. CONCLUSION: The i.p. administration of allantoin may have a beneficial effect on nerve healing.
Asunto(s)
Alantoína/uso terapéutico , Procesamiento de Imagen Asistido por Computador/métodos , Regeneración Nerviosa/efectos de los fármacos , Purinas/uso terapéutico , Nervio Ciático/efectos de los fármacos , Neuropatía Ciática/tratamiento farmacológico , Alantoína/metabolismo , Alantoína/farmacología , Animales , Electromiografía/métodos , Masculino , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Traumatismos de los Nervios Periféricos/patología , Purinas/metabolismo , Purinas/farmacología , Ratas , Ratas Wistar , Nervio Ciático/fisiología , Neuropatía Ciática/patologíaRESUMEN
Soybean (Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.