RESUMEN
The mechanisms underlying regeneration of the central nervous system (CNS) following lesions have been studied extensively in both vertebrate and invertebrate models. To shed light on regeneration, ascidians, a sister group of vertebrates and with remarkable ability to regenerate their brains, constitute an appropriate model system. Glial cells have been implicated in regeneration in vertebrates; however, their role in the adult ascidian CNS regeneration is unknown. A model of degeneration and regeneration using the neurotoxin 3-acetylpyridine (3AP) in the brain of the ascidian Styela plicata was used to identify astrocyte-like cells and investigate their role. We studied the CNS of control ascidians (injected with artificial sea water) and of ascidians whose CNS was regenerating (1 and 10 days after the injection with 3AP). Our results show that the mRNA of the ortholog of glutamine synthetase (GS), a glial-cell marker in vertebrates, is increased during the early stages of regeneration. Confirming the identity of GS, the protein was identified via immunostaining in a cell population during the same regeneration stage. Last, a single ortholog of GS (GSII) is present in ascidian and amphioxus genomes, while two types exist in fungi, some invertebrates, and vertebrates, suggesting that ascidians have lost the GSI type. Taken together, our findings revealed that a cell population expressing glial-cell markers may play a role in regeneration in adult ascidians. This is the first report of astrocyte-like cells in the adult ascidian CNS, and contributes to understanding of the evolution of glial cells among metazoans.
Asunto(s)
Astrocitos , Sistema Nervioso Central , Glutamato-Amoníaco Ligasa , Urocordados , Animales , Urocordados/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/fisiología , Astrocitos/fisiología , Astrocitos/metabolismo , Astrocitos/citología , Glutamato-Amoníaco Ligasa/metabolismo , Regeneración Nerviosa/fisiologíaRESUMEN
Evidence indicates that transcranial direct current stimulation (tDCS) provides therapeutic benefits in different situations, such as epilepsy, depression, inflammatory and neuropathic pain. Despite the increasing use of tDCS, its cellular and molecular basis remains unknown. Astrocytes display a close functional and structural relationship with neurons and have been identified as mediators of neuroprotection in tDCS. Considering the importance of hippocampal glutamatergic neurotransmission in nociceptive pathways, we decided to investigate short-term changes in the hippocampal astrocytes of rats subjected to tDCS, evaluating specific cellular markers (GFAP and S100B), as well as markers of astroglial activity; glutamate uptake, glutamine synthesis by glutamine synthetase (GS) and glutathione content. Data clearly show that a single session of tDCS increases the pain threshold elicited by mechanical and thermal stimuli, as evaluated by von Frey and hot plate tests, respectively. These changes involve inflammatory and astroglial neurochemical changes in the hippocampus, based on specific changes in cell markers, such as S100B and GS. Alterations in S100B were also observed in the cerebrospinal fluid of tDCS animals and, most importantly, specific functional changes (increased glutamate uptake and increased GS activity) were detected in hippocampal astrocytes. These findings contribute to a better understanding of tDCS as a therapeutic strategy for nervous disorders and reinforce the importance of astrocytes as therapeutic targets.
Asunto(s)
Epilepsia , Estimulación Transcraneal de Corriente Directa , Ratas , Animales , Astrocitos/metabolismo , Hipocampo/metabolismo , Epilepsia/metabolismo , Ácido Glutámico/metabolismo , Glutamato-Amoníaco Ligasa/metabolismoRESUMEN
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
Asunto(s)
Glutamato-Amoníaco Ligasa , Ácido Peroxinitroso , Procesamiento Proteico-Postraduccional , Humanos , Cromatografía Liquida , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/farmacología , Espectrometría de Masas en Tándem , Tirosina/metabolismo , Activación Enzimática/efectos de los fármacos , Oxidación-Reducción , Mutación , Agregación Patológica de Proteínas/inducido químicamenteRESUMEN
Anorexia nervosa (AN) is an eating disorder characterized by self-starvation and excessive weight loss with a notorious prevalence in young women. The neurobiology of AN is unknown but murine models, like dehydration induced anorexia (DIA), reproduce weight loss and avoidance of food despite its availability. Astrocytes are known to provide homeostatic support to neurons, but it is little explored if anorexia affects this function. In this study, we tested if DIA disrupts glutamate-glutamine homeostasis associated with astrocytes in the prefrontal cortex (PFC) of young female rats. Our results showed that anorexia reduced the redox state, as well as endogenous glutamate and glutamine. These effects correlated with a reduced expression of the glutamate transporters (GLT-1 and GLAST) and glutamine synthetase, all of them are preferentially expressed by astrocytes. Accordingly, the expression of GFAP was reduced. Anorexia reduced the astrocyte density, promoted a de-ramified morphology, and augmented the de-ramified/ramified astrocyte ratio in the medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC), but not in the motor cortex (M2). The increase of a de-ramified phenotype correlated with increased expression of vimentin and nestin. Based on these results, we conclude that anorexia disrupts glutamate-glutamine homeostasis and the redox state associated with astrocyte dysfunction.
Asunto(s)
Anorexia/metabolismo , Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Homeostasis , Corteza Prefrontal/metabolismo , Animales , Femenino , Glutamato-Amoníaco Ligasa/metabolismo , Nestina , Neuronas/metabolismo , RatasRESUMEN
Bioavailable nitrogen is the limiting nutrient for most agricultural food production. Associative diazotrophs can colonize crop roots and fix their own bioavailable nitrogen from the atmosphere. Wild-type (WT) associative diazotrophs, however, do not release fixed nitrogen in culture and are not known to directly transfer fixed nitrogen resources to plants. Efforts to engineer diazotrophs for plant nitrogen provision as an alternative to chemical fertilization have yielded several strains that transiently release ammonia. However, these strains suffer from selection pressure for nonproducers, which rapidly deplete ammonia accumulating in culture, likely limiting their potential for plant growth promotion (PGP). Here we report engineered Azospirillum brasilense strains with significantly extend ammonia production lifetimes of up to 32 days in culture. Our approach relies on multicopy genetic redundancy of a unidirectional adenylyltransferase (uAT) as a posttranslational mechanism to induce ammonia release via glutamine synthetase deactivation. Testing our multicopy stable strains with the model monocot Setaria viridis in hydroponic monoassociation reveals improvement in plant growth promotion compared to single copy strains. In contrast, inoculation of Zea mays in nitrogen-poor, nonsterile soil does not lead to increased PGP relative to WT, suggesting strain health, resource competition, or colonization capacity in soil may also be limiting factors. In this context, we show that while engineered strains fix more nitrogen per cell compared to WT strains, the expression strength of multiple uAT copies needs to be carefully balanced to maximize ammonia production rates and avoid excessive fitness defects caused by excessive glutamine synthetase shutdown.
Asunto(s)
Amoníaco/metabolismo , Azospirillum brasilense/fisiología , Plantas/metabolismo , Plantas/microbiología , Simbiosis/fisiología , Glutamato-Amoníaco Ligasa/metabolismo , Nitrógeno/metabolismo , Fijación del Nitrógeno/fisiología , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Suelo , Zea mays/metabolismo , Zea mays/microbiologíaRESUMEN
Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.
Asunto(s)
Amoníaco/metabolismo , Azospirillum brasilense/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Setaria (Planta)/microbiología , Azospirillum brasilense/genética , Azospirillum brasilense/crecimiento & desarrollo , Feofitinas/metabolismo , Procesamiento Proteico-Postraduccional , Setaria (Planta)/crecimiento & desarrollo , SimbiosisRESUMEN
BACKGROUND Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania braziliensis, commonly found in Brazil and associated with cutaneous and visceral forms of this disease. Like other organisms, L. braziliensis has an enzyme called glutamine synthetase (LbGS) that acts on the synthesis of glutamine from glutamate. This enzyme plays an essential role in the metabolism of these parasites and can be a potential therapeutic target for treating this disease. OBJECTIVES Investigate LbGS structure and generate structural models of the protein. METHODS We use the method of crosslinking mass spectrometry (XLMS) and generate structural models in silico using I-TASSER. FINDINGS 42 XLs peptides were identified, of which 37 are explained in a monomeric model with the other five indicating LbGS dimerization and pentamers interaction region. The comparison of 3D models generated in the presence and absence of XLMS restrictions probed the benefits of modeling with XLMS highlighting the inappropriate folding due to the absence of spatial restrictions. MAIN CONCLUSIONS In conclusion, we disclose the conservation of the active site and interface regions, but also unique features of LbGS showing the potential of XLMS to probe structural information and explore new drugs.
RESUMEN
BACKGROUND: Early seed germination and a functional root system development during establishment are crucial attributes contributing to nutrient competence under marginal nutrient soil conditions. Chenopodium quinoa Willd (Chenopodiaceae) is a rustic crop, able to grow in marginal areas. Altiplano and Coastal/Lowlands are two representative zones of quinoa cultivation in South America with contrasting soil fertility and edaphoclimatic conditions. In the present work, we hypothesize that the ecotypes of Quinoa from Altiplano (landrace Socaire) and from Coastal/Lowland (landrace Faro) have developed differential adaptive responses in order to survive under conditions of low availability of N in their respective climatic zones of Altiplano and Lowlands. In order to understand intrinsic differences for N competence between landraces, seed metabolite profile and germinative capacity were studied. Additionally, in order to elucidate the mechanisms of N uptake and assimilation at limiting N conditions during establishment, germinated seeds of both landraces were grown at either sufficient nitrate (HN) or low nitrate (LN) supply. We studied the photosynthetic performance, protein storage, root morphometrical parameters, activity and expression of N-assimilating enzymes, and the expression of nitrate transporters of roots in plants submitted to the different treatments. RESULTS: Seeds from Socaire landrace presented higher content of free N-related metabolites and faster seed germination rate compared to Faro landrace. Seedlings of both ecotypes presented similar physiological performance at HN supply, but at LN supply their differences were exalted. At LN, Socaire plants showed an increased root biomass (including a higher number and total length of lateral roots), a differential regulation of a nitrate transporter (a NPF6.3-like homologue) belonging to the Low Affinity Transport System (LATS), and an upregulation of a nitrate transporter (a NRT2.1-like homologue) belonging to the High Affinity nitrate Transport System (HATS) compared to Faro. These responses as a whole could be linked to a higher amount of stored proteins in leaves, associated to an enhanced photochemical performance in Altiplano plants, in comparison to Lowland quinoa plants. CONCLUSIONS: These differential characteristics of Socaire over Faro plants could involve an adaptation to enhanced nitrate uptake under the brutal unfavorable climate conditions of Altiplano.
Asunto(s)
Chenopodium quinoa/metabolismo , Nitrógeno/metabolismo , Plantones/metabolismo , Semillas/metabolismo , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Chenopodium quinoa/genética , Chenopodium quinoa/crecimiento & desarrollo , Chile , Ecotipo , Regulación de la Expresión Génica de las Plantas , Germinación , Glutamato-Amoníaco Ligasa/metabolismo , Nitrato-Reductasa/metabolismo , Transportadores de Nitrato , Nitratos/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Plantones/crecimiento & desarrollo , Semillas/fisiologíaRESUMEN
Conyza spp. is among the main weeds reported worldwide. Due to its aggressiveness, such as high seed production and dispersion, and the growing reports of biotypes resistant to glyphosate, paraquat, and other herbicides, different control practices are required. Thus, this study aimed to evaluate the efficacy of herbicides with sequential application of glufosinate in soybean pre-sowing for control of Conyza spp. with indicative of resistant to paraquat. The study was carried out in the field, at Assis Chateaubriand and Palotina, state of Paraná, Brazil, in the 2018/19 season. The experiments were conducted in a randomized block design with four replications. The treatments consisted in application of glyphosate, 2,4-D, saflufenacil, glufosinate, saflufenacil/imazethapyr, diclosulam, paraquat/diuron, paraquat and imazethapyr/flumioxazin, at different combinations, in soybean pre-sowing. Control of Conyza spp., crop injury to soybean plants and variables related to agronomic performance (plant height and yield) were evaluated. All treatments were selective for soybean, which showed stronger crop injury in the presence of diclosulam herbicide, but this did not compromise soybean agronomic performance. In general, control levels were high for the treatments used. Except for paraquat treatments, in the area with the highest frequency of Conyza spp. with indicative of resistant to paraquat, and imazethapyr/flumioxazin treatment in both areas. These control results emphasized the importance of glufosinate in this management system and showed promising results for saflufenacil/imazethapyr.(AU)
A planta daninha Conyza spp. está entre as principais encontradas em todo o mundo. Devido a sua agressividade, como elevada produção e dispersão de sementes e os crescentes relatos de biótipos resistentes ao glyphosate, paraquat e outros herbicidas, faz-se necessário o uso de diferentes ferramentas de manejo. Assim, o objetivo deste estudo foi avaliar a eficácia de herbicidas com aplicação sequencial de glufosinate em pré-semeadura da soja, no controle de Conyza spp. com indicativo de resistência ao paraquat. O estudo foi realizado em campo nos municípios de Assis Chateaubriand e Palotina, estado do Paraná (PR), Brasil, na safra 2018/19. O delineamento experimental utilizado foi o de blocos casualizados, com quatro repetições. Os tratamentos foram compostos pela aplicação de glyphosate, 2,4-D, saflufenacil, glufosinate, saflufenacil/imazethapyr, diclosulam, paraquat/diuron, paraquat e imazethapyr/flumioxazin, em diferentes combinações em pré-semeadura da soja. Foram avaliados controle de Conyza spp., sintomas de injúria nas plantas de soja e variáveis relacionadas ao desempenho agronômico (altura de plantas e produtividade). Todos os tratamentos mostraram-se seletivos a soja. Visualizou-se maiores sintomas de injúria quando se tinha a presença do herbicida diclosulam, mas não resultou em danos ao desempenho agronômico da soja. De forma geral os níveis de controle foram altos para os tratamentos utilizados, em exceção para os tratamentos com paraquat na área que apresentava maior frequência de Conyza spp. com indicativo de resistência a este herbicida, e para o tratamento com imazethapyr/flumioxazin nas duas áreas. Estes resultados de controle enfatizam a importância do glufosinate neste sistema de manejo e demostram resultados promissores para saflufenacil/imazethapyr.(AU)
Asunto(s)
Conyza/efectos de los fármacos , Control de Malezas , Glycine max/efectos de los fármacos , Herbicidas/administración & dosificaciónRESUMEN
Glutamate is the main excitatory amino acid acting at the level of pre and postsynaptic neurons, as well as in glial cells. It is involved in the coordinated modulation of energy metabolism, glutamine synthesis, and ammonia detoxification. The relationship between the functional status of liver and brain has been known for many years. The most widely recognized aspect of this relation is the brain dysfunction caused by acute liver injury that manifests a wide spectrum of neurologic and psychiatric abnormalities. Inflammation, circulating neurotoxins, and impaired neurotransmission have been reported in this pathophysiology. In the present contribution, we report the effect of a hepatotoxic compound like CCl4 on the expression of key proteins involved in glutamate uptake and metabolism as glutamate transporters and glutamine synthetase in mice liver, brain, and cerebellum. Our findings highlight a differential expression pattern of glutamate transporters in cerebellum. A significant Purkinje cells loss, in parallel to an up-regulation of glutamine synthetase, and astrogliosis in the brain have also been noticed. In the intoxicated liver, glutamate transporter 1 expression is up-regulated, in contrast to glutamine synthetase which is reduced in a time-dependent manner. Taken together our results demonstrate that the exposure to an acute CCl4 insult, leads to the disruption of glutamate transporters expression in the liver-brain axis and therefore a severe alteration in glutamate-mediated neurotransmission might be present in the central nervous system.
RESUMEN
ABSTRACT: Conyza spp. is among the main weeds reported worldwide. Due to its aggressiveness, such as high seed production and dispersion, and the growing reports of biotypes resistant to glyphosate, paraquat, and other herbicides, different control practices are required. Thus, this study aimed to evaluate the efficacy of herbicides with sequential application of glufosinate in soybean pre-sowing for control of Conyza spp. with indicative of resistant to paraquat. The study was carried out in the field, at Assis Chateaubriand and Palotina, state of Paraná, Brazil, in the 2018/19 season. The experiments were conducted in a randomized block design with four replications. The treatments consisted in application of glyphosate, 2,4-D, saflufenacil, glufosinate, saflufenacil/imazethapyr, diclosulam, paraquat/diuron, paraquat and imazethapyr/flumioxazin, at different combinations, in soybean pre-sowing. Control of Conyza spp., crop injury to soybean plants and variables related to agronomic performance (plant height and yield) were evaluated. All treatments were selective for soybean, which showed stronger crop injury in the presence of diclosulam herbicide, but this did not compromise soybean agronomic performance. In general, control levels were high for the treatments used. Except for paraquat treatments, in the area with the highest frequency of Conyza spp. with indicative of resistant to paraquat, and imazethapyr/flumioxazin treatment in both areas. These control results emphasized the importance of glufosinate in this management system and showed promising results for saflufenacil/imazethapyr.
RESUMO: A planta daninha Conyza spp. está entre as principais encontradas em todo o mundo. Devido a sua agressividade, como elevada produção e dispersão de sementes e os crescentes relatos de biótipos resistentes ao glyphosate, paraquat e outros herbicidas, faz-se necessário o uso de diferentes ferramentas de manejo. Assim, o objetivo deste estudo foi avaliar a eficácia de herbicidas com aplicação sequencial de glufosinate em pré-semeadura da soja, no controle de Conyza spp. com indicativo de resistência ao paraquat. O estudo foi realizado em campo nos municípios de Assis Chateaubriand e Palotina, estado do Paraná (PR), Brasil, na safra 2018/19. O delineamento experimental utilizado foi o de blocos casualizados, com quatro repetições. Os tratamentos foram compostos pela aplicação de glyphosate, 2,4-D, saflufenacil, glufosinate, saflufenacil/imazethapyr, diclosulam, paraquat/diuron, paraquat e imazethapyr/flumioxazin, em diferentes combinações em pré-semeadura da soja. Foram avaliados controle de Conyza spp., sintomas de injúria nas plantas de soja e variáveis relacionadas ao desempenho agronômico (altura de plantas e produtividade). Todos os tratamentos mostraram-se seletivos a soja. Visualizou-se maiores sintomas de injúria quando se tinha a presença do herbicida diclosulam, mas não resultou em danos ao desempenho agronômico da soja. De forma geral os níveis de controle foram altos para os tratamentos utilizados, em exceção para os tratamentos com paraquat na área que apresentava maior frequência de Conyza spp. com indicativo de resistência a este herbicida, e para o tratamento com imazethapyr/flumioxazin nas duas áreas. Estes resultados de controle enfatizam a importância do glufosinate neste sistema de manejo e demostram resultados promissores para saflufenacil/imazethapyr.
RESUMEN
To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.(AU)
RESUMEN
ABSTRACT To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.
Asunto(s)
Carbono/metabolismo , Cloruro de Sodio/metabolismo , Vigna/metabolismo , Nitrógeno/metabolismo , Microbiología del Suelo , Cloruro de Sodio/análisis , Actinobacteria/fisiología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Bradyrhizobium/fisiología , Inoculantes Agrícolas/fisiología , Vigna/crecimiento & desarrollo , Vigna/microbiología , Aminoácidos/metabolismo , Fijación del NitrógenoRESUMEN
To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.
Asunto(s)
Carbono/metabolismo , Nitrógeno/metabolismo , Cloruro de Sodio/metabolismo , Vigna/metabolismo , Actinobacteria/fisiología , Inoculantes Agrícolas/fisiología , Aminoácidos/metabolismo , Bradyrhizobium/fisiología , Fijación del Nitrógeno , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Estrés Salino , Cloruro de Sodio/análisis , Microbiología del Suelo , Vigna/crecimiento & desarrollo , Vigna/microbiologíaRESUMEN
Nitrogen (N) can alleviate metal toxicity. However, as of yet, there have been no studies showing the efficacy of NO3-/NH4+ in mitigating Cu toxicity. The objective of this study was to evaluate the Cu toxicity on the nutritional and productive attributes of Panicum maximum cv. Tanzania as well as the role of NO3- and NH4+ ratios in nutritional homeostasis. The experiment was conducted using 3 × 4 factorial treatments arranged in a randomized complete block design with three replicates. The treatments were three NO3-/NH4+ ratios (100/0, 70/30, and 50/50) and four Cu rates (0.3, 250, 500, and 1000 µmol L-1) in nutrient solution. Copper concentrations in the diagnostic leaves (DL) were highest in plants grown under 70/30 NO3-/NH4+ ratios and a Cu rate of 1000 µmol L-1. In this combination, it was observed that DL had higher concentrations of NH4+, greater glutamine synthetase activity, lower chlorophyll concentration (SPAD value), and lower shoot dry mass, suggesting high disorders of nutritional homeostasis. Plants receiving N in the form of NO3- and 1000 Cu µmol L-1 showed that DL had lower concentrations of Cu, higher concentration of chlorophyll, higher NO3- concentration, higher nitrate reductase activity, and higher NO3- accumulation in the roots, suggesting a reduction in disorders of nutritional homeostasis. The disorders on mineral uptake, N assimilation, and biomass production caused by Cu toxicity are shown to be affected by NO3-/NH4+ ratios, and N supply via NO3- allowed for better homeostasis of the forage grass.
Asunto(s)
Amoníaco/análisis , Cobre/toxicidad , Homeostasis/efectos de los fármacos , Nitratos/análisis , Poaceae/crecimiento & desarrollo , Contaminantes del Suelo/toxicidad , Biomasa , Clorofila/metabolismo , Monitoreo del Ambiente/métodos , Poaceae/efectos de los fármacos , TanzaníaRESUMEN
INTRODUCTION AND AIM: Hepatic encephalopathy (HE), caused by hyperammonemia resulting from liver disease, is a spectrum of neuropsychiatric and motor disorders that can lead to death. Existing therapies are deficient and alternative treatments are needed. We have shown that gene therapy with a baculovirus vector containing the glutamine synthetase (Bac-GS) gene is efficient for reducing ammonia levels in an acute hyperammonemia rat model. However, the most common condition resulting from liver disease is chronic hyperammonemia. In this work, Bac-GS was evaluated in bile-duct ligated rats, a chronic liver disease model with hyperammonemia and some characteristics of Type C HE. MATERIAL AND METHODS: Bac-GS was tested for mediating GS overexpression in HeLa cells and H9C2 myotubes. For determining the utility of Bac-GS for the reduction of ammonia levels in a chronic hyperammonemia animal model, four groups of rats were treated: control, sham, ligated with Bac-GS and ligated with Bac-GFP. Baculoviruses were injected i.m. 18 days post-surgery. Blood was drawn 2, 3 and 4 weeks post-surgery and plasma ammonia concentrations were quantified. RESULTS: In protein lysates of cells and myotubes transduced with Bac-GS, a 44 kDa band corresponding to GS was detected. Significant results were obtained in the hyperammonemic bile-duct ligated rat model, as plasma ammonia was reduced to normal levels 3 days after treatment with Bac-GS. Furthermore, a transitory effect of Bac-GS was observed. CONCLUSION: Our results show that gene therapy by delivering GS is a promising alternative for treatment of hyperammonemia in acute-on-chronic liver failure patients with HE.
Asunto(s)
Baculoviridae/genética , Terapia Genética/métodos , Encefalopatía Hepática/etiología , Encefalopatía Hepática/terapia , Hiperamonemia/complicaciones , Análisis de Varianza , Animales , Western Blotting , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Vectores Genéticos , Glutamato-Amoníaco Ligasa/administración & dosificación , Células HeLa/citología , Células HeLa/patología , Encefalopatía Hepática/patología , Humanos , Hiperamonemia/fisiopatología , Distribución Aleatoria , Ratas , Factores de Riesgo , Sensibilidad y EspecificidadRESUMEN
Glutamate is the major excitatory transmitter of the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed both in neurons and in glial cells. Recent evidence has shown that glutamate uptake systems, particularly enriched in glia cells, trigger biochemical cascades in a similar fashion as receptors. A tight regulation of glutamate extracellular levels prevents neuronal overstimulation and cell death, and it is critically involved in glutamate turnover. Glial glutamate transporters are responsible of the majority of the brain glutamate uptake activity. Once internalized, this excitatory amino acid is rapidly metabolized to glutamine via the astrocyte-enriched enzyme glutamine synthetase. A coupling between glutamate uptake and glutamine synthesis and release has been commonly known as the glutamate/glutamine shuttle. Taking advantage of the established model of cultured Bergmann glia cells, in this contribution, we explored the gene expression regulation of glutamine synthetase. A time- and dose-dependent regulation of glutamine synthetase protein and activity levels was found. Moreover, glutamate exposure resulted in the transient shift of glutamine synthetase mRNA from the monosomal to the polysomal fraction. These results demonstrate a novel mode of glutamate-dependent glutamine synthetase regulation and strengthen the notion of an exquisite glia neuronal interaction in glutamatergic synapses.
Asunto(s)
Glutamato-Amoníaco Ligasa/metabolismo , Ácido Glutámico/metabolismo , Neuroglía/enzimología , Biosíntesis de Proteínas , Animales , Células Cultivadas , Embrión de Pollo , Glutamato-Amoníaco Ligasa/genética , Modelos Biológicos , Polirribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Despite that astrocytes and microglia do not communicate by electrical impulses, they can efficiently communicate among them, with each other and with neurons, to participate in complex neural functions requiring broad cell-communication and long-lasting regulation of brain function. Glial cells express many receptors in common with neurons; secrete gliotransmitters as well as neurotrophic and neuroinflammatory factors, which allow them to modulate synaptic transmission and neural excitability. All these properties allow glial cells to influence the activity of neuronal networks. Thus, the incorporation of glial cell function into the understanding of nervous system dynamics will provide a more accurate view of brain function. Our current knowledge of glial cell biology is providing us with experimental tools to explore their participation in neural network modulation. In this chapter, we review some of the classical, as well as some recent, pharmacological tools developed for the study of astrocyte's influence in neural function. We also provide some examples of the use of these pharmacological agents to understand the role of astrocytes in neural network function and dysfunction.
Asunto(s)
Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Metionina Sulfoximina/farmacología , Red Nerviosa/efectos de los fármacos , Oligopéptidos/farmacología , Aconitato Hidratasa/antagonistas & inhibidores , Aconitato Hidratasa/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Caprilatos/farmacología , Comunicación Celular/efectos de los fármacos , Citratos/farmacología , Fluoroacetatos/farmacología , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Red Nerviosa/citología , Red Nerviosa/metabolismo , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transmisión SinápticaRESUMEN
Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by D-[(3)H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc(-) system inhibitor) decreased OGD-induced D-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing D-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.