RESUMEN
Background: Glutamine synthetase (GS), glutamate synthase (GOGAT), and nitrate reductase (NR) are key enzymes involved in nitrogen assimilation and metabolism in plants. However, the systematic analysis of these gene families lacked reports in soybean (Glycine max (L.) Merr.), one of the most important crops worldwide. Methods: In this study, we performed genome-wide identification and characterization of GS, GOGAT, and NR genes in soybean under abiotic and nitrogen stress conditions. Results: We identified a total of 10 GS genes, six GOGAT genes, and four NR genes in the soybean genome. Phylogenetic analysis revealed the presence of multiple isoforms for each gene family, indicating their functional diversification. The distribution of these genes on soybean chromosomes was uneven, with segmental duplication events contributing to their expansion. Within the nitrogen assimilation genes (NAGs) group, there was uniformity in the exon-intron structure and the presence of conserved motifs in NAGs. Furthermore, analysis of cis-elements in NAG promoters indicated complex regulation of their expression. RT-qPCR analysis of seven soybean NAGs under various abiotic stresses, including nitrogen deficiency, drought-nitrogen, and salinity, revealed distinct regulatory patterns. Most NAGs exhibited up-regulation under nitrogen stress, while diverse expression patterns were observed under salt and drought-nitrogen stress, indicating their crucial role in nitrogen assimilation and abiotic stress tolerance. These findings offer valuable insights into the genomic organization and expression profiles of GS, GOGAT, and NR genes in soybean under nitrogen and abiotic stress conditions. The results have potential applications in the development of stress-resistant soybean varieties through genetic engineering and breeding.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Glycine max , Nitrógeno , Filogenia , Glycine max/genética , Glycine max/metabolismo , Nitrógeno/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Estrés Fisiológico/genética , Glutamato Sintasa/genética , Glutamato Sintasa/metabolismo , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Genoma de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cromosomas de las Plantas/genética , SequíasRESUMEN
INTRODUCTION: Although valproic acid is generally well tolerated, hepatotoxicity is a common side effect in patients receiving long-term treatment. However, the mechanisms underlying valproic acid-associated hepatotoxicity remain elusive. METHODS: To investigate the mechanisms and explore the potential risk factors for valproic acid-associated hepatotoxicity, 165 age-matched pediatric patients were recruited for laboratory tests and glutamate-glutamine cycle analysis. RESULTS: The concentration of glutamate in patients with hepatotoxicity was significantly greater than that in control patients, while the concentration of glutamine in patients with hepatotoxicity was significantly lower than that in control patients (P <0.05). In addition, the frequencies of the heterozygous with one mutant allele and homozygous with two mutant alleles genotypes in glutamate-ammonia ligase rs10911021 were significantly higher in the hepatotoxicity group than those in the control group (47.1 percent versus 32.5 percent, P = 0.010; 17.6 percent versus 5.2 percent, P = 0.001, respectively). Moreover, heterozygous carriers with one mutant allele and homozygous carriers with two mutant alleles genotypes of glutamate-ammonia ligase rs10911021 exhibited significant differences in the concentrations of glutamine and glutamate concentrations (P Ë 0.001 and P = 0.001, respectively) and liver function indicators (activities of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase, P <0.001, respectively). Furthermore, logistic regression analysis indicated that glutamate-ammonia ligase rs10911021 (P = 0.002, odds ratio: 3.027, 95 percent confidence interval, 1.521 - 6.023) and glutamate (P = 0.001, odds ratio: 2.235, 95 percent confidence interval, 1.369 - 3.146) were associated with a greater risk for hepatotoxicity, while glutamine concentrations were negatively associated with hepatotoxicity (P = 0.001, odds ratio: 0.711, 95 percent confidence interval, 0.629 - 0.804). DISCUSSION: Understanding pharmacogenomic risks for valproic acid induced hepatotoxicity might help direct patient specific care. Limitations of our study include the exclusive use of children from one location and concomitant medication use in many patients. CONCLUSION: Perturbation of the glutamate-glutamine cycle is associated with valproic acid-associated hepatotoxicity. Moreover, glutamate-ammonia ligase rs10911021, glutamate and glutamine concentrations are potential risk factors for valproic acid-associated hepatotoxicity.
Asunto(s)
Anticonvulsivantes , Enfermedad Hepática Inducida por Sustancias y Drogas , Epilepsia , Glutamato-Amoníaco Ligasa , Ácido Glutámico , Glutamina , Ácido Valproico , Humanos , Ácido Valproico/efectos adversos , Femenino , Ácido Glutámico/metabolismo , Masculino , Niño , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Glutamato-Amoníaco Ligasa/genética , Anticonvulsivantes/efectos adversos , Preescolar , Factores de Riesgo , Adolescente , Estudios de Casos y Controles , GenotipoRESUMEN
Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.
Asunto(s)
Cloroplastos , Glutamato-Amoníaco Ligasa , Hemípteros , Insectos Vectores , Oryza , Phytoplasma , Hojas de la Planta , Animales , Hemípteros/microbiología , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/genética , Phytoplasma/fisiología , Hojas de la Planta/microbiología , Hojas de la Planta/metabolismo , Oryza/microbiología , Oryza/genética , Insectos Vectores/microbiología , Cloroplastos/metabolismo , Enfermedades de las Plantas/microbiología , Clorofila/metabolismo , Plantas Modificadas Genéticamente , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
Glutamine synthetase (GS) is a key enzyme involved in nitrogen assimilation and the maintenance of C/N balance, and it is strictly regulated in all bacteria. In cyanobacteria, GS expression is controlled by nitrogen control A (NtcA) transcription factor, which operates global nitrogen regulation in these photosynthetic organisms. Furthermore, posttranslational regulation of GS is operated by protein-protein interaction with GS inactivating factors (IFs). In this study, we describe an additional regulatory mechanism involving an antisense RNA. In Nostoc sp. PCC 7120, the gifA gene (encoding GS inactivating factor IF7) is transcribed downstream of the GS (glnA) gene, from the opposite strand, and the gifA mRNA extends into the glnA coding sequence in antisense orientation. Therefore, the dual RNA transcript that encodes gifA constitutes two functional regions: a 5' protein-coding region, encoding IF7, and a 3' untranslated region that acts as an antisense to glnA. By increasing the levels of such antisense RNA either in cis or in trans, we demonstrate that the amount of GS activity can be modulated by the presence of the antisense RNA. The tail-to-tail disposition of the glnA and gifA genes observed in many cyanobacterial strains from the Nostocales clade suggests the prevalence of such antisense RNA-mediated regulation of GS in this group of cyanobacteria.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Glutamato-Amoníaco Ligasa , Nostoc , ARN sin Sentido , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , ARN sin Sentido/genética , Nostoc/genética , Nostoc/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
Extensively applied glufosinate (GLU) will trigger molecular alterations in nontarget tea plants (Camellia sinensis), which inadvertently disturbs metabolites and finally affects tea quality. The mechanistic response of tea plants to GLU remains unexplored. This study investigated GLU residue behavior, the impact on photosynthetic capacity, specialized metabolites, secondary pathways, and transcript levels in tea seedlings. Here, GLU mainly metabolized to MPP and accumulated more in mature leaves than in tender ones. GLU catastrophically affected photosynthesis, leading to leaf chlorosis, and decreased Fv/Fm and chlorophyll content. Physiological and biochemical, metabolomics, and transcriptomics analyses were integrated. Showing that GLU disrupted the photosynthetic electron transport chain, triggered ROS and antioxidant system, and inhibited photosynthetic carbon fixation. GLU targeted glutamine synthetase (GS) leading to the accumulation of ammonium and the inhibition of key umami L-theanine, causing a disorder in nitrogen metabolism, especially for amino acids synthesis. Interestingly, biosynthesis of primary flavonoids was sacrificed for defensive phenolic acids and lignin formulation, leading to possible losses in nutrition and tenderness in leaves. This study revealed the defense intricacies and potential quality deterioration of tea plants responding to GLU stress. Valuable insights into detoxification mechanisms for non-target crops post-GLU exposure were offered.
Asunto(s)
Aminobutiratos , Camellia sinensis , Fotosíntesis , Hojas de la Planta , Camellia sinensis/genética , Camellia sinensis/metabolismo , Camellia sinensis/efectos de los fármacos , Aminobutiratos/toxicidad , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/genética , Estrés Fisiológico , Metabolómica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Plantones/efectos de los fármacos , Plantones/metabolismo , Herbicidas/toxicidad , Multiómica , GlutamatosRESUMEN
The enzyme glutamine synthetase (GLN) is mainly responsible for the assimilation and reassimilation of nitrogen (N) in higher plants. Although the GLN gene has been identified in various plants, there is little information about the GLN family in cotton (Gossypium spp.). To elucidate the roles of GLN genes in cotton, we systematically investigated and characterized the GLN gene family across four cotton species (G. raimondii, G. arboreum, G. hirsutum, and G. barbadense). Our analysis encompassed analysis of members, gene structure, cis-element, intragenomic duplication, and exploration of collinear relationships. Gene duplication analysis indicated that segmental duplication was the primary driving force for the expansion of the GhGLN gene family. Transcriptomic and quantitative real-time reverse-transcription PCR (qRT-PCR) analyses indicated that the GhGLN1.1a gene is responsive to N induction treatment and several abiotic stresses. The results of virus-induced gene silencing revealed that the accumulation and N use efficiency (NUE) of cotton were affected by the inactivation of GhGLN1.1a. This study comprehensively analyzed the GhGLN genes in Gossypium spp., and provides a new perspective on the functional roles of GhGLN1.1a in regulating NUE in cotton.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa , Gossypium , Nitrógeno , Proteínas de Plantas , Duplicación de Gen , Genes de Plantas , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Gossypium/genética , Gossypium/metabolismo , Familia de Multigenes , Nitrógeno/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield. This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake. While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants. These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Germinación , Glutamato-Amoníaco Ligasa , Semillas , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/enzimología , Semillas/crecimiento & desarrollo , Semillas/genética , Semillas/enzimología , Germinación/genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosol/enzimología , Citosol/metabolismo , Nitrógeno/metabolismo , Plantas Modificadas Genéticamente , Isoenzimas/genética , Isoenzimas/metabolismoRESUMEN
Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.
Asunto(s)
Epilepsia Generalizada , Glutamato-Amoníaco Ligasa , Glutamina , Animales , Humanos , Ratones , Encéfalo/metabolismo , Epilepsia Generalizada/genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamatos/metabolismo , Glutamina/genética , Glutamina/metabolismoRESUMEN
The production of glutamine (Gln) from NO3- and NH4+ requires ATP, reducing power, and carbon skeletons. Plants may redirect these resources to other physiological processes using Gln directly. However, feeding Gln as the sole nitrogen (N) source has complex effects on plants. Under optimal concentrations, Arabidopsis (Arabidopsis thaliana) seedlings grown on Gln have similar primary root lengths, more lateral roots, smaller leaves, and higher amounts of amino acids and proteins compared to those grown on NH4NO3. While high levels of Gln accumulate in Arabidopsis seedlings grown on Gln, the expression of GLUTAMINE SYNTHETASE1;1 (GLN1;1), GLN1;2, and GLN1;3 encoding cytosolic GS1 increases and expression of GLN2 encoding chloroplastic GS2 decreases. These results suggest that Gln has distinct effects on regulating GLN1 and GLN2 gene expression. Notably, Arabidopsis seedlings grown on Gln have an unexpected gene expression profile. Compared with NH4NO3, which activates growth-promoting genes, Gln preferentially induces stress- and defense-responsive genes. Consistent with the gene expression data, exogenous treatment with Gln enhances disease resistance in Arabidopsis. The induction of Gln-responsive genes, including PATHOGENESIS-RELATED1, SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1, WRKY54, and WALL ASSOCIATED KINASE1, is compromised in salicylic acid (SA) biosynthetic and signaling mutants under Gln treatments. Together, these results suggest that Gln may partly interact with the SA pathway to trigger plant immunity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Glutamina , Raíces de Plantas , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/efectos de los fármacos , Glutamina/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Estrés Fisiológico/genética , Resistencia a la Enfermedad/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/efectos de los fármacos , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/genéticaRESUMEN
The glutamine synthetase/glutamic acid synthetase (GS/GOGAT) cycle plays important roles in N metabolism, growth, development, and stress resistance in plants. Excess ammonium (NH4+) restricts growth, but GS can help to alleviate its toxicity. In this study, the 84K model clone of hybrid poplar (Populus alba × P. tremula var. glandulosa), which has reduced biomass accumulation and leaf chlorosis under high-NH4+ stress, showed less severe symptoms in transgenic lines overexpressing GLUTAMINE SYNTHETASE 1;2 (GS1;2-OE), and more severe symptoms in RNAi lines (GS1;2-RNAi). Compared with the wild type, the GS1;2-OE lines had increased GS and GOGAT activities and higher contents of free amino acids, soluble proteins, total N, and chlorophyll under high-NH4+ stress, whilst the antioxidant and NH4+ assimilation capacities of the GS1;2-RNAi lines were decreased. The total C content and C/N ratio in roots and leaves of the overexpression lines were higher under stress, and there were increased contents of various amino acids and sugar alcohols, and reduced contents of carbohydrates in the roots. Under high-NH4+ stress, genes related to amino acid biosynthesis, sucrose and starch degradation, galactose metabolism, and the antioxidant system were significantly up-regulated in the roots of the overexpression lines. Thus, overexpression of GS1;2 affected the carbon and amino acid metabolism pathways under high-NH4+ stress to help maintain the balance between C and N metabolism and alleviate the symptoms of toxicity. Modification of the GS/GOGAT cycle by genetic engineering is therefore a potential strategy for improving the NH4+ tolerance of cultivated trees.
Asunto(s)
Compuestos de Amonio , Carbono , Glutamato-Amoníaco Ligasa , Nitrógeno , Plantas Modificadas Genéticamente , Populus , Populus/genética , Populus/metabolismo , Populus/enzimología , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/genética , Nitrógeno/metabolismo , Carbono/metabolismo , Compuestos de Amonio/metabolismo , Compuestos de Amonio/toxicidad , Plantas Modificadas Genéticamente/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.IMPORTANCEKnowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections-most often caused by Escherichia coli-growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.
Asunto(s)
Escherichia coli , Transcripción Genética , Escherichia coli/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Amoníaco , Glutamina/genética , Urea , Genes BacterianosRESUMEN
The study of astrocytes and its role in the development and evolution of neurodegenerative diseases, including Alzheimer's disease (AD) is essential to fully understand their aetiology. The aim if this study is to deepen into the concept of the heterogeneity of astrocyte subpopulations in the EC and in particular the identification of differentially functioning astrocyte subpopulations that respond differently to AD progression. S100ß protein belongs to group of small calcium regulators of cell membrane channels and pumps that are expressed by astrocytes and is hypothesised to play and have a relevant role in AD development. We analysed the selective differentiation of S100ß-positive astrocytes into Glutamine synthetase (GS) and Glial fibrillary acidic protein (GFAP)-positive sub-groups in the entorhinal cortex (EC) of AD triple transgenic animal model (3xTg-AD). EC is the brain region earliest affected in humans AD but also in this closest animal model regarding their pathology and time course. We observed no changes in the number of S100ß-positive astrocytes between 1 and 18 months of age in the EC of 3xTg-AD mice. However, we identified relevant morphological changes in S100ß/GFAP positive astrocytes showing a significant reduction in their surface and volume whilst an increase in number and percentage. Furthermore, the percentage of S100ß/GS positive astrocyte population was also increased in 18 months old 3xTg-AD mice compared to the non-Tg mice. Our findings reveal the presence of differentially controlled astrocyte populations that respond differently to AD progression in the EC of 3xTg-AD mice. These results highpoints the major astrocytic role together with its early and marked affection in AD and arguing in favour of its importance in neurogenerative diseases and potential target for new therapeutic approaches.
Asunto(s)
Enfermedad de Alzheimer , Animales , Humanos , Lactante , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Corteza Entorrinal/metabolismo , Corteza Entorrinal/patología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Ratones TransgénicosRESUMEN
Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.
Asunto(s)
Diabetes Mellitus Experimental , Niacinamida , Ratas , Masculino , Animales , Niacinamida/farmacología , Niacinamida/metabolismo , NAD/metabolismo , NAD/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Ratas Wistar , Inhibidores de Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Arginasa/genética , Arginasa/metabolismo , Arginasa/farmacología , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamato-Amoníaco Ligasa/farmacología , Estrés Oxidativo , Hígado/metabolismo , Urea/metabolismo , Urea/farmacologíaRESUMEN
Improving nitrogen use efficiency (NUE) is one of the main ways of increasing plant productivity through genetic engineering. The modification of nitrogen (N) metabolism can affect the hormonal content, but in transgenic plants, this aspect has not been sufficiently studied. Transgenic birch (Betula pubescens) plants with the pine glutamine synthetase gene GS1 were evaluated for hormone levels during rooting in vitro and budburst under outdoor conditions. In the shoots of the transgenic lines, the content of indoleacetic acid (IAA) was 1.5-3 times higher than in the wild type. The addition of phosphinothricin (PPT), a glutamine synthetase (GS) inhibitor, to the medium reduced the IAA content in transgenic plants, but it did not change in the control. In the roots of birch plants, PPT had the opposite effect. PPT decreased the content of free amino acids in the leaves of nontransgenic birch, but their content increased in GS-overexpressing plants. A three-year pot experiment with different N availability showed that the productivity of the transgenic birch line was significantly higher than in the control under N deficiency, but not excess, conditions. Nitrogen availability did not affect budburst in the spring of the fourth year; however, bud breaking in transgenic plants was delayed compared to the control. The IAA and abscisic acid (ABA) contents in the buds of birch plants at dormancy and budburst depended both on N availability and the transgenic status. These results enable a better understanding of the interaction between phytohormones and nutrients in woody plants.
Asunto(s)
Betula , Glutamato-Amoníaco Ligasa , Betula/genética , Betula/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Nitrógeno/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The glutamine synthetase (GS) facilitates cancer cell growth by catalyzing de novo glutamine synthesis. This enzyme removes ammonia waste from the liver following the urea cycle. Since cancer development is associated with dysregulated urea cycles, there has been no investigation of GS's role in ammonia clearance. Here, we demonstrate that, although GS expression is increased in the setting of ß-catenin oncogenic activation, it is insufficient to clear the ammonia waste burden due to the dysregulated urea cycle and may thus be unable to prevent cancer formation. In vivo study, a total of 165 male Swiss albino mice allocated in 11 groups were used, and liver cancer was induced by p-DAB. The activity of GS was evaluated along with the relative expression of mTOR, ß-catenin, MMP-14, and GS genes in liver samples and HepG2 cells using qRT-PCR. Moreover, the cytotoxicity of the NH3 scavenger phenyl acetate (PA) and/or GS-inhibitor L-methionine sulfoximine (MSO) and the migratory potential of cells was assessed by MTT and wound healing assays, respectively. The Swiss target prediction algorithm was used to screen the mentioned compounds for probable targets. The treatment of the HepG2 cell line with PA plus MSO demonstrated strong cytotoxicity. The post-scratch remaining wound area (%) in the untreated HepG2 cells was 2.0%. In contrast, the remaining wound area (%) in the cells treated with PA, MSO, and PA + MSO for 48 h was 61.1, 55.8, and 78.5%, respectively. The combination of the two drugs had the greatest effect, resulting in the greatest decrease in the GS activity, ß-catenin, and mTOR expression. MSO and PA are both capable of suppressing mTOR, a key player in the development of HCC, and MMP-14, a key player in the development of HCC. PA inhibited the MMP-14 enzyme more effectively than MSO, implying that PA might be a better way to target HCC as it inhibited MMP-14 more effectively than MSO. A large number of abnormal hepatocytes (5%) were found to be present in the HCC mice compared to mice in the control group as determined by the histopathological lesions scores. In contrast, PA, MSO, and PA + MSO showed a significant reduction in the hepatic lesions score either when protecting the liver or when treating the liver. The molecular docking study indicated that PA and MSO form a three-dimensional structure with NF-κB and COX-II, blocking their ability to promote cancer and cause gene mutations. PA and MSO could be used to manipulate GS activities to modulate ammonia levels, thus providing a potential treatment for ammonia homeostasis.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Masculino , Ratones , Animales , Neoplasias Hepáticas/patología , Carcinoma Hepatocelular/patología , beta Catenina/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Amoníaco/metabolismo , Amoníaco/uso terapéutico , Nitrógeno/uso terapéutico , Metaloproteinasa 14 de la Matriz , Simulación del Acoplamiento Molecular , Serina-Treonina Quinasas TOR , Homeostasis , Urea/uso terapéuticoRESUMEN
Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.
A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.