Increase In Glutamine Synthetase Activity Research Articles

Impacts of linseed oil diet on anxiety and memory extinction after early life stress: A sex-specific analysis of mitochondrial dysfunction, astrocytic markers, and inflammation in the amygdala

Early exposure to stressors affects how the organism reacts to stimuli, its emotional state throughout life, and how it deals with emotional memories. Consequently, it may affect susceptibility to psychopathology later in life. We used an animal model of early stress by maternal separation to study its potential impact on the extinction of aversive memories and anxiety-like behavior in adulthood, as well as its effects on mitochondrial functionality, inflammatory and astrocytic markers in the amygdala. We also assessed whether a diet enriched with linseed oil, known for its high content in omega-3 fats, could be used to attenuate the behavioral and neurochemical effects of early stress. Litters of Wistar rats were divided into controls (intact) or subjected to maternal separation (MS). They were subdivided into two groups receiving isocaloric diets enriched in soy or linseed oils at weaning. In adulthood, the animals were exposed to the open field and the elevated plus maze, to evaluate exploratory activity and anxiety-like behavior. They were also trained in a context of fear conditioning, and afterward subjected to an extinction session, followed by a test session to evaluate the extinction memory. Amygdalae were evaluated for inflammatory cytokines (interleukin (IL)-1beta, IL-6, and tumor-necrose factor (TNF)-alpha), mitochondrial functionality, and astrocyte markers (glial fibrillary acidic protein − GFAP, S100B, and glutamine synthetase activity). MS induced anxiety-like behavior in the elevated plus-maze, which was reversed by a diet enriched in linseed oil offered from weaning. When testing the memory of an extinction session of fear conditioning, MS animals showed more freezing behavior. MS males receiving a linseed oil-enriched diet had lower functional mitochondria in the amygdala. In addition, MS led to increased inflammatory cytokines, particularly IL-1beta, and the diet enriched in linseed oil further increased these levels in MS animals. MS also increased S100B levels. These results point to a higher emotionality presented by MS animals, with higher levels of inflammatory cytokines and S100B. While a diet enriched in linseed oil attenuated anxiety-like behavior, it further altered amygdala IL-1beta and reduced mitochondria functionality, particularly in males. MS also increased glutamine synthetase activity in the amygdala, and this effect was higher when the animals received a diet enriched in linseed oil, particularly in females. In conclusion, these results point to MS effects on emotional behavior, and neurochemical alterations in the amygdala, with sex-specific effects. Although a diet enriched in linseed oil appears to be able to reverse some of MS behavioral effects, these results must be considered with caution, since biochemical parameters could be worsened in MS animals receiving a linseed oil-enriched diet. This knowledge is important for the understanding of mechanisms of action of strategies aiming to reverse early stress effects, and future studies are warranted to determine possible interventions to promote resilience.

24-epibrassinolide enhances drought tolerance in grapevine (Vitis vinifera L.) by regulating carbon and nitrogen metabolism.

Exogenous application of 24-epibrassinolide can alleviate oxidative damage, improve photosynthetic capacity, and regulate carbon and nitrogen assimilation, thus improving the tolerance of grapevine (Vitis vinifera L.) to drought stress. Brassinosteroids (BRs) are a group of plant steroid hormones in plants and are involved in regulating plant tolerance to drought stress. This study aimed to investigate the regulation effects of BRs on the carbon and nitrogen metabolism in grapevine under drought stress. The results indicated that drought stress led to the accumulation of superoxide radicals and hydrogen peroxide and an increase in lipid peroxidation. A reduction in oxidative damage was observed in EBR-pretreated plants, which was probably due to the improved antioxidant concentration. Moreover, exogenous EBR improved the photosynthetic capacity and sucrose phosphate synthase activity, and decreased the sucrose synthase, acid invertase, and neutral invertase, resulting in improved sucrose (190%) and starch (17%) concentrations. Furthermore, EBR pretreatment strengthened nitrate reduction and ammonium assimilation. A 57% increase in nitrate reductase activity and a 13% increase in glutamine synthetase activity were observed in EBR pretreated grapevines. Meanwhile, EBR pretreated plants accumulated a greater amount of proline, which contributed to osmotic adjustment and ROS scavenging. In summary, exogenous EBR enhanced drought tolerance in grapevines by alleviating oxidative damage and regulating carbon and nitrogen metabolism.

Astroglial Alterations in the Hippocampus of Rats Submitted to a Single Trans-Cranial Direct Current Stimulation Trial.

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.

Antibiotics induced changes in nitrogen metabolism and antioxidative enzymes in mung bean (Vigna radiata)

Excessive use and release of antibiotics into the soil environment in the developing world have resulted in altered soil processes affecting terrestrial organisms and posing a serious threat to crop growth and productivity. The present study investigated the influence of exogenously applied oxytetracycline (OXY) and levofloxacin (LEV) on plant physiological responses, key enzymes involved in nitrogen metabolism (e.g., nitrate reductase, glutamine synthetase), nitrogen contents and oxidative stress response of mung bean (Vigna radiata). Plants were irrigated weekly with antibiotics containing water for exposing the plants to different concentrations i.e., 1, 10, 20, 50, and 100 mg L−1. Results showed a significant decrease in nitrate reductase activity in both antibiotic treatments and their mixtures and increased antioxidant enzymatic activities in plants. At lower concentrations of antibiotics (≤20 mg L−1), 53.9 % to 78.4 % increase in nitrogen content was observed in levofloxacin and mixtures compared to the control, resulting in an increase in the overall plant biomass. Higher antibiotic (≥50 mg L−1) concentration showed 58 % decrease in plant biomass content and an overall decrease in plant nitrogen content upon exposure to the mixtures. This was further complemented by 22 % to 42 % increase in glutamine synthetase activity observed in the plants treated with levofloxacin and mixtures. The application of low doses of antibiotics throughout the experiments resulted in lower toxicity symptoms in the plants. However, significantly higher malondialdehyde (MDA) concentrations at higher doses (20 mg L−1 and above) than the control showed that plants' tolerance against oxidative stress was conceded with increasing antibiotic concentrations. The toxicity trend was: levofloxacin > mixture > oxytetracycline.

Alleviation of iron deficiency in pear by ammonium nitrate and nitric oxide

BackgroundIron is essential for the growth and development of trace elements in plants, and iron deficiency can lead to leaf chlorosis. Ammonium and nitrate are the major forms of nitrogen present in soils. Ammonium nitrate alleviates the chlorosis of leaves caused by iron deficiency, but the mechanism is not clear in pear.ResultsAmmonium nitrate induced the increase of nitric oxide (NO) under iron deficiency. We further analyzed the effect of NO by exogenous NO treatment. The results showed that ammonium nitrate and NO increased the activity of ferric chelate reductase. NO induced the expression of multiple IRT genes and promoted the transmembrane transport of irons. Ammonium nitrate and NO promoted the activity of nitrogen assimilation-related enzymes and the nitrogen absorption capacity, and they also increased glutamine synthetase activity. Finally, ammonium nitrate and NO increased chlorophyll synthesis, with subsequent increase in the photosynthetic capacity of plants and accumulation of biomass.ConclusionAmmonium nitrate indirectly alleviates the symptoms of plant yellowing by promoting the increase of NO, which increases the response of iron transporters. Both substances increase the nitrogen accumulation in plants. This study demonstrates a new option for minimizing Fe deficiency by regulating the balance between nutrients.

Nitrogen metabolism plays a major role in the adaptation of the halophytic forage species Sulla carnosa to water deficit and upon stress recovery

Our study aimed to evaluate the changes in the nitrogen compounds and enzyme activities in a forage halophytic species, Sulla carnosa, subjected to water deficit stress and recovery. Seedlings were individually cultivated for two months under two irrigation modes: 100% and 33% of field capacity. Our results showed that water deficit stress induced a strong decline in both leaf biomass production (-70%) and leaf water content. Besides, S. carnosa plants exposed to water deficit stress preferentially allocated photoassimilates to roots while showing a decrease in leaf osmotic potential. Moreover under water deficit conditions, leaf ammonium content remained unchanged, whereas a significant increase in leaf nitrate content was observed. Water deficit stress also triggered an accumulation of free amino acids, mainly proline and asparagine (respectively 18-and 70-fold increases as compared to controls), suggesting their potential role in osmotic adjustment. Despite its increase in leaves of stressed plants, glycine did not exceed 2% of the amino acid pool. The accumulation of glutamate, as a consequence of a high glutamine synthetase (GS) activity and the marked amino acid accumulation in leaves strongly suggest that glutamate turnover is of adaptive significance for S. carnosa, confirming the involvement of glutamate metabolism in the accumulation of adaptive metabolites in plant response to stress conditions. After rehydration, a partial growth re-establishment was observed in S. carnosa, and was concomitant with a better tissue hydration and a decrease in leaf nitrate content, involving a significant increase in GS activity, and a decrease in glutamate dehydrogenase (GDH) deamination activity. Overall, S. carnosa is able to conserve the functional integrity of nitrogen-related metabolic pathways after exposure to stressful conditions.

Increased glutamine synthetase by overexpression of TaGS1 improves grain yield and nitrogen use efficiency in rice

Improving nitrogen use efficiency (NUE) has been a focal point for crop growth and yield throughout the world. Glutamine synthetase (GS), which plays a fundamental role in N metabolism, has been exploited to improve crop NUE. However, increased GS activity in rice by overexpressing its own GS genes hasn't shown superior plant productivity. Here, transgenic rice plants with increased GS activity by overexpressing TaGS1 were analyzed under field and culture conditions at two N rates. Transgenic expression of TaGS1 significantly increases GS activity in leaves, junctions and roots of rice plants relative to wide-type plants. When rice plants grown under consecutive field trials with N rates of 60 and 240 kg/ha, three transgenic lines have higher grain yield than wild-type plants, with increment of 15%–22% in T2 generation and with that of 28%–36% in T3 generation, respectively. And increased panicle numbers (effective tiller numbers) mainly contribute to the advantage of grain yield in transgenic plants. Analysis of N use-related traits shows that transgenic plants with enhanced GS activity promote root capacity to obtain N, N accumulation during growth stages and N remobilization to grains, ultimately conferring 31%–40% improvement of NUE relative to wild-type rice plants.

Glutamine synthetase regulation by dexamethasone, RU486, and compound A in astrocytes derived from aged mouse cerebral hemispheres is mediated via glucocorticoid receptor.

Glucocorticoids (GCs) regulate astrocyte function, while glutamine synthetase (GS), an enzyme highly expressed in astrocytes, is one of the most remarkable GCs-induced genes. GCs mediate their effects through their cognate glucocorticoid receptor (GRα and GRβ isoforms); however, the mechanism via which these isoforms regulate GS activity in astrocytes remains unknown. We used dexamethasone (DEX), a classical GRα/GRβ agonist, RU486, which is a specific GRβ ligand, and Compound A, a known "dissociated" ligand, to delineate the mechanism via which GR modulates GS activity. Aged Mouse Cerebral Hemisphere astrocytes were treated with DEX (1μM), RU486 (1nM-1μM) or compound A (10μM), alone or in combination with DEX. GS activity and expression, GR isoforms (mRNA and protein levels), and GRα subcellular trafficking were measured. DEX increased GS activity in parallel with GRα nuclear translocation. RU486 increased GS activity in absence of GRα nuclear translocation implicating thus a role of GRβ-mediated mechanism compound A had no effect on GS activity implicating a GRα-GRE-mediated mechanism. None of the compounds affected whole-cell GRα protein content. DEX reduced GRα and GRβ mRNA levels, while RU486 increased GRβ gene expression. We provide evidence that GS activity, in astrocytes, is regulated via GRα- and GRβ-mediated pathways with important implications in pathological conditions in which astrocytes are involved.

Does inoculation with associative bacteria improve tolerance to nitrogen deficiency in seedlings of Neotropical tree species?

Nitrogen (N) deficiency generally affects plant physiology, influencing N metabolism and inhibiting photosynthesis and plant growth. The association with plant growth-promoting rhizobacteria (PGPR) can mitigate environmental stresses, such as N deficiency. No studies have examined whether inoculation with PGPR may counterbalance the effects of N restriction on the metabolism and growth of neotropical tree seedlings with different resource-use strategies. The current study aimed to determine the effects of inoculation with PGPR on the tolerance of seedlings of tree species submitted to N deficiency. Two species with acquisitive (Cecropia pachystachya and Heliocarpus popayanensis) and conservative (Cariniana estrellensis and Cabralea canjerana) resource-use strategies were chosen. The strains Ab-V5, of Azospirillum brasilense, and ZK, of Bacillus velezensis, were selected as PGPR for inoculation. Seedlings of the four plant species were grown in a mixture of soil and sand with (N+) or without (N-) N supplementation. The seedlings were also inoculated with Ab-V5 or ZK strains and, as a control, for each N treatment, some of the seedlings were not inoculated with PGPR (Nin). Thus, we assessed the effects of N deficiency, inoculation, and the interaction between these two factors on N metabolism, photosynthetic, and growth variables. Nitrogen deficiency limited photosynthesis in all plant species, in addition to decreasing the concentration of amino acids and proteins and the activity of enzymes of N metabolism. Only the growth of C. canjerana was not inhibited by N restriction. Inoculation with Ab-V5 led to increased growth of C. pachystachya seedlings, which was more pronounced under N supplementation (+70 % of total dry mass). The association with PGPR decreased nitrate reductase activity (-52 %) and increased glutamine synthetase activity (+370 %) in the leaves, among other changes in N metabolism. The growth of H. popayanensis and C. estrellensis seedlings was not affected by inoculation, but metabolic variables in these species were influenced by inoculation with ZK. Photosynthesis increased with inoculation with ZK in H. popayanensis and with Ab-V5 in C. estrellensis. In C. canjerana, both PGPR promoted seedling growth (+11 % of total dry mass). Nitrogen deficiency and inoculation with PGPR influence photosynthesis, growth, and N metabolism of seedlings of tree species that occur in the semideciduous seasonal forest. Moreover, the effects of inoculation are influenced by N supplementation during cultivation. The positive effects of PGPR on growth were higher in N-supplemented seedlings. Regardless of the functional group, inoculation with PGPR of seedlings of tree species of the Atlantic biome stimulates growth and development in optimal N conditions and increases tolerance to N restriction.

Urea- Versus Ammonium Nitrate–Based Fertilizers for Green Sugarcane Cultivation

This study tested the hypothesis that enhanced-efficiency fertilizers, such as urea treated with NBPT, are a feasible substitute for ammonium nitrate–based fertilizers for green sugarcane cultivation. The experimental design included six N sources applied over sugarcane straw at two sites in Central-South Brazil. Treatments included ammonium nitrate, urea, and four urea-based fertilizers treated with different rates of the urease inhibitor N-(n-butyl)thiophosphoric triamide (NBPT) at two rates of N (50 and 100 kg ha−1), plus an additional control. Nitrogen status in the plant, the activity of glutamine synthetase (GS) and nitrate reductase (NR) in the leaves, and sugarcane stalk yield and quality were evaluated. At site 1, N rates increased the SPAD index, stalk yield, N uptake, and sugar yield. At site 2, N rates increased GS activity, N content, the SPAD index, and N uptake, but stalk and sugar yield were not increased due to large variability in these parameters. All N sources resulted in similar yields at both sites. These results were compared with those in the literature. Among 27 studies surveyed, the average sugarcane yield was 97.8 or 97.4 Mg ha−1 under ammonium nitrate or urea fertilization, respectively, and these averages did not differ by the Kruskal-Wallis test. The results of this study demonstrate that enhanced-efficiency fertilizers have the potential to reduce NH3 emissions from urea-based fertilizers in green sugarcane fields without affecting sugarcane yield.

Effects of Olive Mill Wastewater and Two Natural Extracts as Nitrification Inhibitors on Activity of Nitrifying Bacteria, Soil Nitrate Leaching Loss, and Nitrogen Metabolism of Celery (Apium graveolens L.)

Minimizing nitrification of fertilizer ammonium (NH4+) can reduce nitrate (NO3−) contamination of groundwater and increase nitrogen (N) use efficiency (NUE). Olive mill wastewater (OMW), hydroalcoholic extracts of Mentha piperita L. (Mp) and Artemisia annua L. (Aa), and synthetic nitrification inhibitor (NI) dicyandiamide (DCD) were investigated. All NIs reduced activity of nitrifying bacteria and NO3− leached and increased efficiency of N metabolism of celery (Apium graveolens L.) during 56 days in a soil mixture fertilized with ammonium sulfate [(NH4)2SO4]. Soil NO3− leaching losses of DCD, OMW, Mp, and Aa treatments were 24%, 26%, 67%, and 78% of the untreated control loss, respectively, at 35 and 56 days after planting (DAP). Decreased nitrification by NIs resulted in greater concentrations of soil NH4+ correlated with less nitrate reductase (NR) activity in roots and leaves and less soil acidification compared to the control. At 35 and 56 DAP, DCD and OMW treatments decreased NR and increased glutamine synthetase activities in leaves and roots, compared to the control. NIs increased leaf and root protein and amino acids. OMW significantly decreased leaching loss of NO3− to 10% of fertilizer N applied to the soil, compared to 38% of applied NH4+-N leached from the control. OMW proved an effective alternative to DCD to improve NUE of NH4+-fertilizers.

Yeast two-hybrid screening reveals a dual function for the histone acetyltransferase GcnE by controlling glutamine synthesis and development in Aspergillus fumigatus.

The acetyltransferase GcnE is part of the SAGA complex which regulates fungal gene expression through acetylation of chromatin. Target genes of the histone acetyltransferase GcnE include those involved in secondary metabolism and asexual development. Here, we show that the absence of GcnE not only abrogated conidiation, but also strongly impeded vegetative growth of hyphae in the human pathogenic fungus Aspergillus fumigatus. A yeast two-hybrid screen using a Saccharomyces cerevisiae strain whose tRNA molecules were specifically adapted to express A. fumigatus proteins identified two unprecedented proteins that directly interact with GcnE. Glutamine synthetase GlnA as well as a hypothetical protein located on chromosome 8 (GbpA) were identified as binding partners of GcnE and their interaction was confirmed in vivo via bimolecular fluorescence complementation. Phenotypic characterization of gbpA and glnA deletion mutants revealed a role for GbpA during conidiogenesis and confirmed the central role of GlnA in glutamine biosynthesis. The increase of glutamine synthetase activity in the absence of GcnE indicated that GcnE silences GlnA through binding. This finding suggests an expansion of the regulatory role of GcnE in A. fumigatus.

TRAP1 Inhibition Increases Glutamine Synthetase Activity in Glutamine Auxotrophic Non-small Cell Lung Cancer Cells.

Cancer cells are distinct in terms of glutamine dependence. Here we investigated the different susceptibility of glutamine-independent and glutamine-dependent non-small cell lung cancer (NSCLC) to treatment with tumor necrosis factor receptor-associated protein 1 (TRAP1) inhibitor gamitrinib-triphenylphosphonium (G-TPP). Cell viability and proliferation under glutamine deprivation and G-TPP treatment were determined by the MTT and colony-formation assays. Protein and mRNA expression were determined by western blot and quantitative polymerase chain reaction. Colorimetric-based assay was performed to check for glutamine synthetase (GS) activity. NSCLC cells showed diverse adaptation under glutamine-depleted condition and were categorized into glutamine-independent and glutamine-dependent cells. Treatment with G-TPP particularly increased GS activity and induced cell death due to energy shortage indicated by phosphorylated AMP-activated protein kinase (AMPK) in glutamine-dependent cells. This finding provides better understanding of TRAP1-mediated glutamine metabolism through GS activity, and evidence that TRAP1 could be a promising therapeutic target for glutamine-addicted cancer.

A195 GLUTAMINE SYNTHETASE IN ENDOTHELIAL CELLS OF THE BLOOD-BRAIN BARRIER: NEW TARGET FOR THE TREATMENT OF HEPATIC ENCEPHALOPATHY?

The liver plays a major role in regulating ammonia levels in the blood. Therefore, in liver disease the loss of hepatic function leads to hyperammonemia and increased brain ammonia and consequently hepatic encephalopathy (HE). Ammonia-lowering strategies remain the mainstay therapeutic strategy. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which in the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (primarily in astrocytes) but has never been thoroughly explored in the BBB. Therefore, the aim is to evaluate GS expression in endothelial cells of the BBB. Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS expression by western blot using brain lysates as positive control and by immunohistochemistry (co-localized with caveolin-1, a marker for ECs). In addition, to understand the effect of ammonia on GS, ECs were exposed to 1mM of ammonia chloride for 48h. Finally, ECs were subjected to plasma from bile-duct ligated (BDL) rats (model of chronic liver disease) or sham-operated controls. We have characterized this BDL model and found both systemic oxidative stress and inflammation, in addition to hyperammonemia. ECs expressed GS mRNA, protein and activity. However, expression of GS was lower compared to brain lysate control samples (p<0.05). GS expression in CMV showed similar results to brain but GS activity was less (p<0.05). Using immunohistochemistry, GS was detected in ECs and in vessels of brain from naïve rats. When cells were submitted to ammonia, an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS was decreased when compared to sham-operated controls (p<0.01). These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme compared to that found in the brain, could explain why GS has never been reported in these cells. Interestingly, ammonia stimulates GS in ECs, but its activity is decreased in the presence of other pathogenic factors in plasma from cirrhotic rats such as oxidative stress and inflammation. We speculate that a downregulation of GS allows for a faster and easier entry of ammonia into the brain and therefore may be implicated in the pathogenesis of HE. We anticipate increasing GS in ECs of the BBB could become a new therapeutic target for HE. CIHR

Recombination between DEP1 and NRT1.1B under japonica and indica genetic backgrounds to improve grain yield in rice

DEP1 and NRT1.1B genes for improving nitrogen utilization efficiency (NUE) have been widely used in japonica and indica rice (Oryza sativa L.) breeding, respectively. The aim of this study was to determine the effect of the non-allelic recombination of two genes on agronomic traits, grain yield and NUE under japonica and indica genetic backgrounds. One hundred and fifty-one recombinant inbred lines were derived from a cross between japonica variety SN265 with dep1/NRT1.1B alleles and indica variety R99 with DEP1/nrt1.1b alleles. Among these, we found four genotypes (DEP1/NRT1.1B, DEP1/nrt1.1b, dep1/NRT1.1B and dep1/nrt1.1b) under japonica and indica genetic backgrounds. Results showed a significant increase in nitrate reductase activity, N uptake and grain yield under low N growth condition, after the nrt1.1b allele was introduced into japonica lines with dep1. Whereas, the introduction of dep1 allele into indica lines with nrt1.1b increased glutamine synthetase activity, N transfer and grain yield under both low and high N growth conditions. Our results showed diverse physiological pathway between dep1 and nrt1.1b alleles in the N metabolism of rice, providing new strategies in flexible application of dep1 and nrt1.1b alleles for improvement of NUE in japonica and indica rice breeding.

Ameliorative effects of melatonin on dark-induced leaf senescence in gardenia (Gardenia jasminoides Ellis): leaf morphology, anatomy, physiology and transcriptome

Cut gardenia (Gardenia jasminoides Ellis) foliage is widely used as a vase material or flower bouquet indoors; however, insufficient indoor light accelerates its senescence, which shortens its viewing time. In this study, applying melatonin to delay gardenia leaf senescence when exposed to extremely low light condition (darkness), and the results showed that 1.0 mM was the effective concentration. At this concentration, chlorophyll contents and chlorophyll fluorescence parameters (Fv/Fm, Fv/F0 and Y(II)) increased, while the carotenoid and flavonoid contents decreased. Meanwhile, stress physiological indices decreased in response to exogenous melatonin application, whereas an increase in glutamine synthetase activity, water and soluble protein contents was observed. Moreover, exogenous melatonin application also reduced leaf programmed cell death under darkness, increased the endogenous melatonin level, expression levels of tryptophan decarboxylase gene, superoxide dismutase and catalase activities and the ascorbate–glutathione cycle, and maintained more intact anatomical structures. Furthermore, transcriptome sequencing revealed that various biological processes responded to exogenous melatonin application, including carbohydrate metabolism, amino acid metabolism, lipid metabolism, plant hormone signal transduction and pigment biosynthesis. Consequently, dark-induced leaf senescence in gardenia was significantly delayed. These results provided a better understanding for improving the ornamental value of cut gardenia foliage using melatonin.

Acute effects of phenylbutyrate on glutamine, branched-chain amino acid and protein metabolism in skeletal muscles of rats.

Phenylbutyrate (PB) acts as chemical chaperone and histone deacetylase inhibitor, which is used to decrease ammonia in urea cycle disorders and has been investigated for use in the treatment of a number of lethal illnesses. We performed invivo and invitro experiments to examine the effects of PB on glutamine (GLN), branched-chain amino acid (BCAA; valine, leucine and isoleucine) and protein metabolism in rats. In the first study, animals were sacrificed one hour after three injections of PB (300mg/kg b.w.) or saline. In the second study, soleus (SOL, slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles were incubated in a medium with or without PB (5mM). L-[1-14 C] leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. PB treatment decreased GLN, BCAA and branched-chain keto acids (BCKAs) in blood plasma, decreased BCAA and increased GLN concentrations in muscles, and increased GLN synthetase activities in muscles. Addition of PB to incubation medium increased leucine oxidation (55% in EDL, 29% in SOL), decreased BCKA and increased GLN in medium of both muscles, increased GLN in muscles, decreased protein synthesis in SOL and increased proteolysis in EDL. It is concluded that PB decreases BCAA, BCKA and GLN in blood plasma, activates BCAA catabolism and GLN synthesis in muscle and exerts adverse effects on protein metabolism. The results indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be a useful prospective agent which could be effective in management of maple syrup urine disease.

The interactive effect of digesting a meal and thermal acclimation on maximal enzyme activities in the gill, kidney, and intestine of goldfish (Carassius auratus).

Surrounding environmental temperatures affect many aspects of ectotherm physiology. Generally, organisms can compensate at one or more biological levels, or allow temperature to dictate processes such as enzyme activities through kinetic effects on reaction rates. As digestion also alters physiological processes such as enzyme activities, this study determined the interacting effect of thermal acclimation (8 and 20 °C) and digesting a single meal on maximal enzyme activities in three tissues of the goldfish (Carrassius auratus). Acclimation to elevated temperatures decreased branchial Na+, K+, ATPase (NKA) activity. In contrast, acclimation to elevated temperatures had no effect on citrate synthase (CS) or pyruvate kinase (PK) activity in any tissue, nor were renal NKA or glutamine synthetase (GS) activities impacted. Warm water-acclimation exaggerated the positive impact of digestion on intestinal and branchial NKA activities and intestinal GS activity only, but digestion had no effect in the kidney. CS and PK did not display intestinal zonation; however, there was a distinct increase towards the distal intestine in NKA and GS activities. Zonation of NKA was more prominent in warm-acclimated animals, while acclimation temperature did not affect intestinal heterogeneity of GS. Finally, the impact of tissue protein content on enzyme activity was discussed. We conclude that the intestine and gill of warm-acclimated goldfish exhibited an augmented capacity for increasing several enzyme activities in response to digestion while the kidney was unaffected by thermal acclimation or digesting a single meal. However, this amplified capacity was ameliorated by alterations in tissue protein content. Amplified increases in NKA activity may ultimately have implications for ATP demand in these tissues, while increased GS activity may beneficially increase ammonia-detoxifying capacity in the intestine.

Glutamine Synthetase in Endothelial Cells of the Blood–brain Barrier: Less But Not Insignificant?

Background and Aim: The liver plays a major role in regulating ammonia levels in the blood. Therefore, during liver disease, the loss of hepatic function leads to hyperammonemia and consequently increased brain ammonia and hepatic encephalopathy (HE). Reducing ammonia neurotoxicity through systemic ammonia-lowering strategies remains the mainstay therapeutic strategy for HE. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which during the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in the endothelial cells of the BBB. Methods: Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS activity and protein (brain lysate was used as positive control). GS was also evaluated by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, GS activity was assessed in ECs exposed to 1 mM of ammonium chloride for 48 h. Finally, GS activity and protein expression were evaluated in ECs submitted to plasma from 6-week bile-duct ligated (BDL) rats or sham-operated controls for 72 h. Results: ECs expressed mRNA, protein and activity of GS. However, EC's expression (normalized to g/protein) of GS was lower compared to brain lysate control samples (P < 0.05). GS protein expression in CMV showed similar results to that of brain but GS activity was significantly less in CMV (P < 0.05). Using immunohistochemistry, GS was detected in ECs and in CMV from naïve rats. When ECs were exposed to ammonia (1 mM), an increase in GS activity was demonstrated (P < 0.05). However, when exposed to conditioned medium from BDL rats, GS activity and protein expression were decreased when compared to sham-operated controls (P < 0.05). Conclusion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme in CMVs compared to brain, could reason why GS has never been reported in these cells. Interestingly, ammonia stimulates GS activity in ECs, but GS activity is decreased following treatment with plasma from BDL rats. This suggests other factors such as oxidative stress and inflammation, could inhibit GS activity. We speculate that a downregulation of GS in the BBB allows for a faster entry of ammonia into the brain and therefore may play a significant role in the onset of HE. We anticipate upregulating GS in ECs of the BBB could become a new therapeutic target for HE. The authors have none to declare.

Current state of knowledge of hepatic encephalopathy (part I): newer treatment strategies for hyperammonemia in liver failure

Alterations in interorgan metabolism of ammonia play an important role in the onset of hyperammonemia in liver failure. Glutamine synthetase (GS) in muscle is an important target for ammonia removal strategies in hyperammonemia. Ornithine Phenylacetate (OP) is hypothesized to remove ammonia by providing glutamate as a substrate for increased GS activity and hence glutamine production. The newly generated glutamine conjugates with phenylacetate forming phenylacetylglutamine which can be excreted in the urine, providing an excretion pathway for ammonia. We have also shown that OP targets glycine metabolism, providing an additional ammonia reducing effect.