Inhibitor Of Glutamine Synthetase Research Articles

ADSCs-derived exosomes ameliorate hepatic fibrosis by suppressing stellate cell activation and remodeling hepatocellular glutamine synthetase-mediated glutamine and ammonia homeostasis

BackgroundHepatic fibrosis is a common pathologic stage in chronic liver disease development, which might ultimately lead to liver cirrhosis. Accumulating evidence suggests that adipose-derived stromal cells (ADSCs)-based therapies show excellent therapeutic potential in liver injury disease owing to its superior properties, including tissue repair ability and immunomodulation effect. However, cell-based therapy still limits to several problems, such as engraftment efficiency and immunoreaction, which impede the ADSCs-based therapeutics development. So, ADSCs-derived extracellular vesicles (EVs), especially for exosomes (ADSC-EXO), emerge as a promise cell-free therapeutics to ameliorate liver fibrosis. The effect and underlying mechanisms of ADSC-EXO in liver fibrosis remains blurred.MethodsHepatic fibrosis murine model was established by intraperitoneal sequential injecting the diethylnitrosamine (DEN) for two weeks and then carbon tetrachloride (CCl4) for six weeks. Subsequently, hepatic fibrosis mice were administrated with ADSC-EXO (10 μg/g) or PBS through tail vein infusion for three times in two weeks. To evaluate the anti-fibrotic capacity of ADSC-EXO, we detected liver morphology by histopathological examination, ECM deposition by serology test and Sirius Red staining, profibrogenic markers by qRT-PCR assay. LX-2 cells treated with TGF-β (10 ng/ml) for 12 h were conducted for evaluating ADSC-EXO effect on activated hepatic stellate cells (HSCs). RNA-seq was performed for further analysis of the underlying regulatory mechanisms of ADSC-EXO in liver fibrosis.ResultsIn this study, we obtained isolated ADSCs, collected and separated ADSCs-derived exosomes. We found that ADSC-EXO treatment could efficiently ameliorate DEN/CCl4-induced hepatic fibrosis by improving mice liver function and lessening hepatic ECM deposition. Moreover, ADSC-EXO intervention could reverse profibrogenic phenotypes both in vivo and in vitro, including HSCs activation depressed and profibrogenic markers inhibition. Additionally, RNA-seq analysis further determined that decreased glutamine synthetase (Glul) of perivenous hepatocytes in hepatic fibrosis mice could be dramatically up-regulated by ADSC-EXO treatment; meanwhile, glutamine and ammonia metabolism-associated key enzyme OAT was up-regulated and GLS2 was down-regulated by ADSC-EXO treatment in mice liver. In addition, glutamine synthetase inhibitor would erase ADSC-EXO therapeutic effect on hepatic fibrosis.ConclusionsThese findings demonstrated that ADSC-derived exosomes could efficiently alleviate hepatic fibrosis by suppressing HSCs activation and remodeling glutamine and ammonia metabolism mediated by hepatocellular glutamine synthetase, which might be a novel and promising anti-fibrotic therapeutics for hepatic fibrosis disease.

Identification of Concomitant Inhibitors against Glutamine Synthetase and Isocitrate Lyase in Mycobacterium tuberculosis from Natural Sources.

Tuberculosis (T.B.) is a disease that occurs due to infection by the bacterium, Mycobacterium tuberculosis (Mtb), which is responsible for millions of deaths every year. Due to the emergence of multidrug and extensive drug-resistant Mtb strains, there is an urgent need to develop more powerful drugs for inclusion in the current tuberculosis treatment regime. In this study, 1778 molecules from four medicinal plants, Azadirachta indica, Camellia sinensis, Adhatoda vasica, and Ginkgo biloba, were selected and docked against two chosen drug targets, namely, Glutamine Synthetase (G.S.) and Isocitrate Lyase (I.C.L.). Molecular Docking was performed using the Glide module of the Schrӧdinger suite to identify the best-performing ligands; the complexes formed by the best-performing ligands were further investigated for their binding stability via Molecular Dynamics Simulation of 100 ns. The present study suggests that Azadiradione from Azadirachta indica possesses the potential to inhibit Glutamine Synthetase and Isocitrate Lyase of M. tuberculosis concomitantly. The excellent docking score of the ligand and the stability of receptor-ligand complexes, coupled with the complete pharmacokinetic profile of Azadiradione, support the proposal of the small molecule, Azadiradione as a novel antitubercular agent. Further, wet lab analysis of Azadiradione may lead to the possible discovery of a novel antitubercular drug.

Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate

Main conclusionAmplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds.Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha−1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = – 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

Glutamine synthetase plays an important role in ammonium tolerance of Myriophyllum aquaticum

High-strength ammonium (NH4+), the main characteristic of swine wastewater, poses a significant threat to the rural ecological environment. As a novel phytoremediation technology, Myriophyllum aquaticum wetlands have high tolerance and removal rate of NH4+. Glutamine synthetase (GS), a pivotal enzyme in nitrogen (N) metabolism, is hypothesized to play an important role in the tolerance of M. aquaticum to high NH4+. Herein, the responses of M. aquaticum to GS inhibition by 0.1 mM methionine sulfoximine (MSX) under 15 mM NH4+ were investigated. After 5 days, visible NH4+ toxicity symptoms were observed in MSX-treated plants. Compared with the control, the NH4+ accumulation in the leaves increased by 20.99 times, while that of stems and roots increased by 3.27 times and 47.76 %, suggesting that GS inhibition had a greater impact on the leaves. GS inhibition decreased pigments in the leaves by 8.64 %–41.06 %, triggered oxidative stress, and affected ions concentrations in M. aquaticum. The concentrations of glutamine (Gln) and asparagine decreased by 63.46 %–97.43 % and 12.37 %–76.41 %, respectively, while the concentrations of most other amino acids increased after 5 days of MSX treatment, showing that GS inhibition reprogrammed the amino acids synthesis. A decrease in Gln explains the regulations of N-related genes, including increased expression of AMT in roots and decreased expression of GS, GOGAT, GDH, and AS, which would cause further NH4+ accumulation via promoting NH4+ uptake and decreasing NH4+ assimilation in M. aquaticum. This study revealed for the first time that GS inhibition under high NH4+ condition can lead to phytotoxicity in M. aquaticum due to NH4+ accumulation. The physiological and molecular responses of the leaves, stems, and roots confirmed the importance of GS in the high NH4+ tolerance of M. aquaticum. These findings provide new insights into NH4+ tolerance mechanisms in M. aquaticum and a theoretical foundation for the phytoremediation of high NH4+-loaded swine wastewater.

Bisphosphonate Use Is Not Associated With Tuberculosis Risk Among Patients With Osteoporosis: A Nationwide Cohort Study.

Bisphosphonates are considered an effective inhibitor of glutamine synthetase and thus can be used for treating tuberculosis (TB). However, its clinical benefit in TB remains unknown. We conducted a population-based cohort study by using the Taiwan National Health Insurance Research Database and TB databases of the Taiwan Centers for Disease Control. Patients with osteoporosis and a history of bone fracture from 2007 to 2014 were identified. Among them, bisphosphonate users and propensity score-matched nonusers were selected. A stratified multivariable Cox proportional hazard regression model was employed to investigate the independent predictors of TB. Among 218908 patients with osteoporosis and bone fracture, 46842 bisphosphonate users and 46842 propensity score-matched nonusers were selected. Within the 2-year follow-up, 723 patients-348 in the user group and 375 in the nonuser group-developed TB. Bisphosphonate use was not an independent predictor of TB in the multivariable Cox proportional hazard model (adjusted hazard ratio, 0.86; 95%CI, 0.71-1.04); however, male sex, older age, being bedridden, and steroid use were independent risk factors. The real-world data revealed that bisphosphonate use did not protect patients with osteoporosis against TB.

On-Demand Generation of Peroxynitrite from an Integrated Two-Dimensional System for Enhanced Tumor Therapy.

Nanosystem-mediated tumor radiosensitization strategy combining the features of X-ray with infinite penetration depth and high atomic number elements shows considerable application potential in clinical cancer therapy. However, it is difficult to achieve satisfactory anticancer efficacy using clinical radiotherapy for the majority of solid tumors due to the restrictions brought about by the tumor hypoxia, insufficient DNA damage, and rapid DNA repair during and after treatment. Inspired by the complementary advantages of nitric oxide (NO) and X-ray-induced photodynamic therapy, we herein report a two-dimensional nanoplatform by the integration of the NO donor-modified LiYF4:Ce scintillator and graphitic carbon nitride nanosheets for on-demand generation of highly cytotoxic peroxynitrite (ONOO-). By simply adjusting the Ce3+ doping content, the obtained nanoscintillator can realize high radioluminescence, activating photosensitive materials to simultaneously generate NO and superoxide radical for the formation of ONOO- in the tumor. Obtained ONOO- effectively amplifies therapeutic efficacy of radiotherapy by directly inducing mitochondrial and DNA damage, overcoming hypoxia-associated radiation resistance. The level of glutamine synthetase (GS) is downregulated by ONOO-, and the inhibition of GS delays DNA damage repair, further enhancing radiosensitivity. This work establishes a combinatorial strategy of ONOO- to overcome the major limitations of radiotherapy and provides insightful guidance to clinical radiotherapy.

Interaction between 4-hydroxyphenylpyruvate dioxygenase–inhibiting and reactive oxygen species–generating herbicides for the control of annual weed species in corn

AbstractThe complementary modes of action of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors have been credited for the synergistic weed control improvement of several species. Recent research discovered that reactive oxygen species (ROS) generation and subsequent lipid peroxidation is the cause of cell death by the glutamine synthetase inhibitor glufosinate. Therefore, a basis for synergy exists between glufosinate and HPPD inhibitors, but the interaction has not been well reported. Four field experiments were conducted in Ontario, Canada, in 2020 and 2021 to determine the interaction between HPPD-inhibiting (mesotrione and tolpyralate) and ROS-generating (atrazine, bromoxynil, bentazon, and glufosinate) herbicides on control of annual weed species in corn (Zea mays L.). The ROS generators were synergistic with the HPPD inhibitors and provided ≥95% control of velvetleaf (Abutilon theophrasti Medik.), except for tolpyralate + glufosinate, which was additive at 8 wk after application (WAA) and provided 87% control. Tank mixes of HPPD inhibitors plus ROS generators were synergistic for the control of common ragweed (Ambrosia artemisiifolia L.), except for tolpyralate + glufosinate, which was antagonistic at 8 WAA. Tolpyralate + glufosinate was antagonistic for the control of barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] and Setaria spp. at 8 WAA. Common lambsquarters (Chenopodium album L.) control at 8 WAA was synergistic and ≥95% with mesotrione plus atrazine, bromoxynil, or glufosinate and with tolpyralate plus bromoxynil or bentazon. Herbicide tank mixes were generally additive for the control of wild mustard (Sinapis arvensis L.) at 8 WAA, except for the synergistic tank mixes of tolpyralate plus atrazine or bromoxynil; however, each tank mix provided 97% to 100% control of S. arvensis. Results from this study demonstrate that co-application of ROS generators with mesotrione or tolpyralate controlled all broadleaf weed species >90% at 8 WAA, with the exceptions of A. artemisiifolia and C. album control with tolpyralate + glufosinate. Mesotrione plus PSII inhibitors controlled E. crus-galli and Setaria spp. 48 to 68 percentage points less than tolpyralate plus the respective PSII inhibitor at 8 WAA; however, mesotrione + glufosinate and tolpyralate + glufosinate controlled the grass weed species similarly.

Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism

Abstract Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve complete and durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are therefore crucial in the effort to improve immunotherapies. One avenue for manipulation of CTLs is through alterations to cellular metabolic programs. T cells radically alter their metabolism upon activation. Prior studies have shown pan-inhibition of glutaminolysis using the inhibitor 6-Diazo-5-oxo-l-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. Herein, I utilize a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – the enzyme responsible for catalyzing the conversion of glutamate to glutamine – conferred a fitness advantage to CTLs. In future experiments, inhibition and genetic deletion of GS will be utilized to investigate the role of GS in CTL activation, proliferation, effector cytokine production, and memory formation.

Тhe role of the glutamatergic system in the mechanism of development of hyperbaric oxygen seizures

The purpose of the scientific article was to study the neurophysiological mechanisms of the occurrence of convulsive activity when breathing oxygen under high pressure under conditions of hyperbaric oxygen and the role of the glutamatergic system in this process.Research methods: Wistar rats, males, weighing 200–250 g. in the amount of 20 were used in the work. The animals were placed in a pressure chamber and compressed with medical oxygen up to 5 ATА. Behavioral reactions and signs of convulsive activity were recorded in animals during compression and during subsequent exposure. Two experimental groups were previously injected with a glutamine synthetase inhibitor at various concentrations 6 hours before compression.The main results showed that irreversible inhibition of glutamine synthetase in the rat brain under the action of increased oxygen pressure led to a significant decrease in the latent period of convulsions.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

Organic fertigation system in saline‐sodic soils: A new paradigm for the restoration of soil health

AbstractA continuous decline in soil organic C (SOC) and an increase in salt‐affected areas are a challenge to future sustainability. We searched on the scientific databases of Web of Science, Scopus (Elsevier) database, and Google. We reviewed 101 studies of the last 25 yr with the following keywords: drip irrigation, fertigation system, liquid biochar, liquid organic, organic fertigation, saline‐sodic soils, and salinization. Our objectives were to identify knowledge gaps to direct future organic research and assess the effect of organic amendments on meeting the plant's N, P, and K requirements in saline‐sodic soils. The review showed that organic fertigation systems reduce salt concentration in irrigation water and inhibit glutamine synthetase by N‐fixing diazotrophs. The system increases photosynthetic activities in plants, microbial biomass C, bacterial colonization, plant–mycorrhizal association, and phospholipid‐derived fatty acids from microbes. It increases essential soil nutrients, including SOC; increases salt/water stress tolerance; and reduces Na+ ion toxicity in plants by regulating abscisic acid pathways in saline‐sodic soils. It creates a conducive environment for phosphatase, dehydrogenase, urease, protease, α‐amylase, and gibberellins. This review sketches the role of silicic acid, salicylic acid, and liquid organics in plant growth mechanisms: enhanced photosynthesis, increased salt/water tolerance, and reduced Na+ toxicity. It suggests novel strategies to develop a task force to promote a community–corporate farming system to adopt cost‐effective green technologies for bioresource regeneration, diverse market production, and social capital generation. We develop a synergy between organic fertigation system and community–corporate farming to bring much‐needed agricultural sustainability.

Saul Brusilow: Understanding and treating diseases of ammonia toxicity

This article reviews the scientific career and accomplishments of the late Dr. Saul Brusilow, Professor of Pediatrics at Johns Hopkins. Dr. Brusilow's career was focused on diseases involving hyperammonemia. He and his colleagues developed a set of drugs that could lower ammonia levels in patients with genetic disorders of the urea cycle by providing alternative pathways for the synthesis of excretable nitrogenous molecules. Those drugs and their derivatives represent one of the earliest and most successful drug therapies for genetic diseases. Turning their attention to brain swelling caused by liver disease, Dr. Brusilow and colleagues developed the Osmotic Gliopathy Hypothesis to help explain the mechanism of ammonia toxicity, postulating that high ammonia drives glutamine synthetase in astrocytes to produce elevated levels of glutamine that act as a potent osmolyte, drawing water into the cell and causing cerebral edema. This hypothesis suggests that inhibiting glutamine synthetase with its well-characterized inhibitor, methionine sulfoximine, might prove therapeutic in cases of hepatic encephalopathy, a conclusion supported by their subsequent studies in animals. But although the drugs developed to treat hyperammonemia resulting from urea cycle disorders were successfully developed and approved by the FDA, the compound suggested as a treatment for hepatic encephalopathy was unable to attract sufficient interest and investment to be tested for use in humans.

Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1β and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Glutamine deficiency induces lipolysis in adipocytes

Glutamine is the most abundant amino acid in the body, and adipose tissue is one of the glutamine-producing organs. Glutamine has important and unique metabolic functions; however, its effects in adipocytes are still unclear. 3T3-L1 adipocytes produced and secreted glutamine dependent on glutamine synthetase, but preadipocytes did not. The inhibition of glutamine synthetase by l-methionine sulfoximine (MSO) impaired the differentiation of preadipocytes to mature adipocytes, and this inhibitory effect of MSO was rescued by exogenous glutamine supplementation. Glutamine concentrations were low, and Atgl gene expression was high in epididymal white adipose tissues of fasting mice in vivo. In 3T3-L1 adipocytes, glutamine deprivation induced Atgl expression and increased glycerol concentration in culture medium. Atgl expression is regulated by FoxO1, and glutamine deprivation reduced FoxO1 phosphorylation (Ser256), indicating the activation of FoxO1. These results demonstrate that glutamine is necessary for the differentiation of preadipocytes and regulates lipolysis through FoxO1 in mature adipocytes.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment

AbstractMechanisms underlying the resistance of acute lymphoblastic leukemia (ALL) blasts to l-asparaginase are still incompletely known. Here we demonstrate that human primary bone marrow mesenchymal stromal cells (MSCs) successfully adapt to l-asparaginase and markedly protect leukemic blasts from the enzyme-dependent cytotoxicity through an amino acid trade-off. ALL blasts synthesize and secrete glutamine, thus increasing extracellular glutamine availability for stromal cells. In turn, MSCs use glutamine, either synthesized through glutamine synthetase (GS) or imported, to produce asparagine, which is then extruded to sustain asparagine-auxotroph leukemic cells. GS inhibition prevents mesenchymal cells adaptation to l-asparaginase, lowers glutamine secretion by ALL blasts, and markedly hinders the protection exerted by MSCs on leukemic cells. The pro-survival amino acid exchange is hindered by the inhibition or silencing of the asparagine efflux transporter SNAT5, which is induced in mesenchymal cells by ALL blasts. Consistently, primary MSCs from ALL patients express higher levels of SNAT5 (P < .05), secrete more asparagine (P < .05), and protect leukemic blasts (P < .05) better than MSCs isolated from healthy donors. In conclusion, ALL blasts arrange a pro-leukemic amino acid trade-off with bone marrow mesenchymal cells, which depends on GS and SNAT5 and promotes leukemic cell survival during l-asparaginase treatment.

Biochemical Basis for the Time-of-Day Effect on Glufosinate Efficacy against Amaranthus palmeri

Glufosinate, a glutamine synthetase (GS) inhibitor, often provides variable weed control depending on environmental conditions such as light, temperature and humidity at the time of application. Midday applications normally provide improved efficacy compared to applications at dawn or dusk. We investigated the biochemical basis for the time-of-day effect on glufosinate efficacy in Amaranthus palmeri. GS1/GS2 gene expression and GS1/GS2 protein abundance were assessed in different parts (young leaves, old leaves, and roots) of plants incubated in the dark compared to those in the light. The turnover of GS total activity was also evaluated overtime following glufosinate treatment at midday compared to dusk application. The results suggest that GS in A. palmeri is less expressed and less abundant in the dark compared to in the light. Midday application of glufosinate under intense light conditions following application provide full control of A. palmeri plants. Consequently, these plants are unable to recover GS activity by de novo protein synthesis. Full activity of GS is required for complete inhibition by the irreversible inhibitor glufosinate. Therefore, glufosinate applications should always be performed in the middle of the day when sunlight is intense, to prevent weed escapes from the herbicide treatment.

Different glutamate sources and endogenous co-agonists activate extrasynaptic NMDA receptors on amacrine cells of the rod pathway microcircuit.

The NMDA receptors (NMDARs) expressed by AII and A17 amacrine cells, the two main inhibitory interneurons of the rod pathway microcircuit in the mammalian retina, are exclusively extrasynaptic, activated by ambient levels of glutamate, and molecularly distinct, with AII and A17 amacrines expressing GluN2B- and GluN2A-containing receptors, respectively. This important sensory microcircuit thus provides a unique model to study the activation and function of extrasynaptic NMDARs. Here, we investigated the sources of glutamate and the endogenous co-agonists (d-serine or glycine) that activate these distinct populations of NMDARs. With acute slices from rat retina, we used whole-cell voltage-clamp recording and measurement of current noise to monitor levels of NMDAR activity. Pre-incubation of retina with bafilomycin A1 (an inhibitor of neurotransmitter uptake into synaptic vesicles) abolished NMDAR-mediated noise in AII, but not A17 amacrines, suggesting a vesicular source of glutamate activates AII NMDARs, whereas a non-vesicular source activates A17 NMDARs. Pre-incubation of retina with l-methionine sulfoximine (an inhibitor of glutamine synthetase) also abolished NMDAR-mediated noise in AII, but not A17 amacrines, suggesting a neuronal source of glutamate activates AII NMDARs, whereas a glial source activates A17 NMDARs. Enzymatic breakdown of d-serine reduced NMDAR-mediated noise in AII, but not A17 amacrines, suggesting d-serine is the endogenous co-agonist at AII, but not A17 NMDARs. Our results reveal unique characteristics of these two populations of extrasynaptic NMDARs. The differential and independent activation of these receptors is likely to provide specific contributions to the signal processing and plasticity of the cellular components of the rod pathway microcircuit.

Network-Related Changes in Neurotransmitters and Seizure Propagation During Rodent Epileptogenesis.

To test the hypothesis that glutamate and GABA are linked to the formation of epilepsy networks and the triggering of spontaneous seizures, we examined seizure initiation/propagation characteristics and neurotransmitter levels during epileptogenesis in a translationally relevant rodent model of mesial temporal lobe epilepsy. The glutamine synthetase (GS) inhibitor methionine sulfoximine was infused into one of the hippocampi in laboratory rats to create a seizure focus. Long-term video-intracranial EEG recordings and brain microdialysis combined with mass spectrometry were used to examine seizure initiation, seizure propagation, and extracellular brain levels of glutamate and GABA. All seizures (n = 78 seizures, n = 3 rats) appeared first in the GS-inhibited hippocampus of all animals, followed by propagation to the contralateral hippocampus. Propagation time decreased significantly from 11.65 seconds early in epileptogenesis (weeks 1-2) to 6.82 seconds late in epileptogenesis (weeks 3-4, paired t test, p = 0.025). Baseline extracellular glutamate levels were 11.6-fold higher in the hippocampus of seizure propagation (7.3 µM) vs the hippocampus of seizure onset (0.63 µM, analysis of variance/Fisher least significant difference, p = 0.01), even though the concentrations of the major glutamate transporter proteins excitatory amino acid transporter subtypes 1 and 2 and xCT were unchanged between the brain regions. Finally, extracellular GABA in the seizure focus decreased significantly from baseline several hours before a spontaneous seizure (paired t test/false discovery rate). The changes in glutamate and GABA suggest novel and potentially important roles of the amino acids in epilepsy network formation and in the initiation and propagation of spontaneous seizures.

Acute-on-chronic liver failure: update on pathogenesis, therapeutic targets, predictive models, and liver transplantation.

Acute-on-chronic liver failure (ACLF) is a clinical syndrome in patients with chronic liver disease that is associated with multiple organ failures and a high short-term mortality. Systemic inflammation is suggested to play a key role in its pathogenesis, although the precise causative mechanism is unknown. The purpose of this review is to present and discuss new findings related to: mechanisms underlying ACLF, therapeutic targets, risk prediction models for developing ACLF, and liver transplantation for ACLF. Recent studies of ACLF pathophysiology classified the immunosuppressive phenotype in monocytes. Investigation of therapeutic strategies identified inhibition of toll-like receptor-4 (TLR-4) and glutamine synthetase (GLUL) as potential targets. Recent studies identified novel risk prediction models for developing ACLF and enhanced our understanding of liver transplantation for ACLF to guide clinicians in determining that patients will benefit from transplantation. Improved knowledge on the pathogenesis of ACLF and identification of TLR-4 and GLUL may lead to clinical trials to study the efficacy of these novel therapeutic targets for patients with ACLF. Liver transplantation is the only current treatment for ACLF. Given the limited availability of donor organs, recent studies have identified ACLF patients who may merit the highest waitlist priority.

Regulation of glutamine synthetase activity by transcriptional and posttranslational modifications negatively influences ganoderic acid biosynthesis in Ganoderma lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.