Glutamate Dehydrogenase Activity Research Articles

Ammonium metabolism in Selaginella bryopteris in response to dehydration-rehydration and characterisation of desiccation tolerant, thermostable, cytosolic glutamine synthetase from plant.

Water deficit (WD) has adverse effects on plant growth, and acclimation requires responses allowing primary metabolism to continue. Resurrection plants can serve as model system to gain insight into metabolic regulation during WD. We herein report the response of a resurrection lycophyte, Selaginella bryopteris, to dehydration-rehydration cycle with emphasis on ammonium metabolism. Dehydration of S. bryopteris fronds resulted in decrease of total protein and increase of free ammonium levels and the effect was reversed on rehydration. The proline content increased twice after 24 h of dehydration, which again recovered to background levels comparable to that at full turgor state. The specific activity of glutamine synthetase (GS) didn't change significantly till 6 h and then declined by 21% after 24 h of dehydration, whereas specific activities of glutamate synthase (GOGAT) and aminating glutamate dehydrogenase (GDH) were enhanced significantly during dehydration. The deaminating activity of GDH also increased during dehydration albeit at a slower rate. Immunoblot analysis indicated overexpression of GS and GDH polypeptides during dehydration and their levels declined on rehydration. The results suggested significant role of GDH along with GS/GOGAT in production of nitrogen-rich amino acids for desiccation tolerance. Unlike higher plants S. bryopteris expressed GS only in cytosol. The enzyme had pH and temperature optima of 5.5 and 60°C, respectively, and it retained 96% activity on preincubation at 60°C for 30 min indicating thermostability. Hence, like higher plants the cytosolic GS from S. bryopteris has a conserved role in stress tolerance.

Changes to hepatic nutrient dynamics and energetics in rainbow trout (Oncorhynchus mykiss) following exposure to and recovery from hydraulic fracturing flowback and produced water

Hydraulic fracturing flowback and produced water (FPW) is a highly complex and heterogenous wastewater by-product of hydraulic fracturing practices. To date, no research has examined how FPW exposure to freshwater biota may affect energetic homeostasis following subsequent induction of detoxification processes. Rainbow trout (Oncorhynchus mykiss) were acutely exposed for 48 h to either 2.5% or 7.5% FPW, and hepatic metabolism was assessed either immediately or following a 3-week recovery period. Induction of xenobiotic metabolism was observed with an 8.8-fold increase in ethoxyresorufin-O-deethylase (EROD) activity after 48 h exposure to 7.5% FPW, alongside a 10.3-fold increase in the mRNA abundance of cyp1a, both of which returned to basal level after three weeks. Glucose uptake capacity was elevated by 6.8- and 12.9-fold following 2.5% and 7.5% FPW exposure, respectively, while alanine uptake was variable. Activity measurements and mRNA abundance of key enzymes involved in hepatic metabolism indicated that aerobic metabolism was maintained with exposure, as was glycolysis. Gluconeogenesis, as measured by phosphoenolpyruvate carboxykinase (PEPCK) activity, decreased by ~30% 48 h following 2.5% FPW exposure and ~20% 3 weeks after 7.5% FPW exposure. The abundance of pepck mRNA activity followed similar, yet non-significant, trends. Finally, a delayed increase in amino acid catabolism was observed, as glutamate dehydrogenase (GDH) activity was increased 2-fold in 7.5% FPW exposed fish when compared to saline control fish at the 3-week time point. We provide evidence to suggest that although hepatic metabolism is altered following acute FPW exposure, metabolic homeostasis generally returns 3-weeks post-exposure.

How to easily detect plant NADH-glutamate dehydrogenase (GDH) activity? A simple and reliable in planta procedure suitable for tissues, extracts and heterologous microbial systems

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems.

Nitric Oxide Signal, Nitrogen Metabolism, and Water Balance Affected by γ-Aminobutyric Acid (GABA) in Relation to Enhanced Tolerance to Water Stress in Creeping Bentgrass.

γ-Aminobutyric acid (GABA) plays an important role in regulating stress tolerance in plants. Purposes of this study was to determine the effect of an exogenous supply of GABA on tolerance to water stress in creeping bentgrass (Agrostis stolonifera), and further reveal the GABA-induced key mechanisms related to water balance, nitrogen (N) metabolism and nitric oxide (NO) production in response to water stress. Plants were pretreated with or without 0.5 mM GABA solution in the roots for 3 days, and then subjected to water stress induced by −0.52 MPa polyethylene glycol 6000 for 12 days. The results showed that water stress caused leaf water deficit, chlorophyll (Chl) loss, oxidative damage (increases in superoxide anion, hydrogen peroxide, malondialdehyde, and protein carbonyl content), N insufficiency, and metabolic disturbance. However, the exogenous addition of GABA significantly increased endogenous GABA content, osmotic adjustment and antioxidant enzyme activities (superoxide dismutase, catalase, dehydroascorbate reductase, glutathione reductase and monodehydroascorbate reductase), followed by effectively alleviating water stress damage, including declines in oxidative damage, photoinhibition, and water and Chl loss. GABA supply not only provided more available N, but also affected N metabolism through activating nitrite reductase and glutamine synthetase activities under water stress. The supply of GABA did not increase glutamate content and glutamate decarboxylase activity, but enhanced glutamate dehydrogenase activity, which might indicate that GABA promoted the conversion and utilization of glutamate for maintaining Chl synthesis and tricarboxylic acid cycle when creeping bentgrass underwent water stress. In addition, GABA-induced NO production, depending on nitrate reductase and NO-associated protein pathways, could be associated with the enhancement of antioxidant defense. Current findings reveal the critical role of GABA in regulating signal transduction and metabolic homeostasis in plants under water-limited condition.

Glutamate dehydrogenase as a biomarker for mitotoxicity; insights from furosemide hepatotoxicity in the mouse.

Glutamate dehydrogenase (GLDH) is a liver-specific biomarker of hepatocellular damage currently undergoing qualification as a drug development tool. Since GLDH is located within the mitochondrial matrix, it has been hypothesized that it might also be useful in assessing mitotoxicity as an initiating event during drug-induced liver injury. According to this hypothesis, hepatocyte death that does not involve primary mitochondrial injury would result in release of intact mitochondria into circulation that could be removed by high speed centrifugation and result in lower GLDH activity measured in spun serum vs un-spun serum. A single prior study in mice has provided some support for this hypothesis. We sought to repeat and extend the findings of this study. Accordingly, mice were treated with the known mitochondrial toxicant, acetaminophen (APAP), or with furosemide (FS), a toxicant believed to cause hepatocyte death through mechanisms not involving mitotoxicity as initiating event. We measured GLDH levels in fresh plasma before and after high speed centrifugation to remove intact mitochondria. We found that both APAP and FS treatments caused substantial hepatocellular necrosis that correlated with plasma alanine aminotransferase (ALT) and GLDH elevations. The plasma GLDH activity in both the APAP- and FS- treated mice was not affected by high-speed centrifugation. Interestingly, the ratio of GLDH:ALT was 5-fold lower during FS compared to APAP hepatotoxicity. Electron microscopy confirmed that both APAP- and FS-treatments had resulted in mitochondrial injury. Mitochondria within vesicles were only observed in the FS-treated mice raising the possibility that mitophagy might account for reduced release of GLDH in the FS-treated mice. Although our results show that plasma GLDH is not clinically useful for evaluating mitotoxicity, the GLDH:ALT ratio as a measure of mitophagy needs to be further studied.

Increased Ammonium Assimilation Activity in the Scleractinian Coral Pocillopora damicornis but Not Its Symbiont After Acute Heat Stress

Ammonium is the main nitrogen resources for scleractinian coral-Symbiodiniaceae symbiotic association, and there is urgent need to investigate the involvement of ammonium assimilation in the heat acclimation of the symbiotic association to heat stress. In the present study, symbiont density and chlorophyll content, as well as redox and ammonium assimilation enzyme activities in two symbiotic partners of the scleractinian coral Pocillopora damicornis were firstly investigated after acute heat stress (32 °C). Symbiont density and chlorophyll content decreased significantly at 24 h (1.81×105 cell cm-2, p < 0.05) and 36 h (23.25 pg cell-1, p < 0.05) after heat stress, respectively. We observed significant activity increases of coral nitric oxide synthase, superoxide dismutase and catalase, and symbiont superoxide dismutase after heat stress, but no alterations in the activities of symbiont nitric oxide synthase and catalase during the whole experiment period. The activities of coral glutamine synthetase and glutamine oxoglutarate aminotransferase began to increase significantly at 24 h, and reached the peak at 36 h after heat stress. As for glutamate dehydrogenase activity, a significant increase was observed only at 36 h after heat stress. The activities of symbiont glutamine synthetase and glutamine oxoglutarate aminotransferase did not change significantly after heat stress. Secondly, symbiont density, chlorophyll content, apoptosis rate, coral total antioxidant capacity, and caspase3 activation level were determined after glufosinate (glutamine synthetase inhibitor) treatment. Symbiont density decreased significantly at 6 h (1.25×106 cell cm-2, p < 0.05) after glufosinate treatment, and symbiont chlorophyll content also decreased significantly during 12-24 h after glufosinate treatment, with the lowest level at 12 h (16.69 pg cell-1, p < 0.05). Furthermore, the coral total antioxidant capacity and caspase3 activation level both increased significantly at 12 h (0.57 U mg-1, 2.08-fold, p < 0.05) after glufosinate treatment, while no significant change was observed for the symbiont apoptosis rate. These results suggest that the ammonium assimilation activity in the coral host P. damicornis, not its symbiont, was induced by acute heat stress, which might contribute to the acclimatization of the symbiotic association to high temperature through regulating coral antioxidant capacity and apoptosis.

Salt Stress Induces Differentiated Nitrogen Uptake and Antioxidant Responses in Two Contrasting Barley Landraces from MENA Region

The interaction between salinity and nitrogen metabolism has been investigated in two barley landraces, one tolerant (“100/1B”) and one susceptible to salinity (“Barley medenine”) from the Middle East and North Africa (MENA) region. Barley plants were exposed to 50 mM NaCl for 7 days; then, salinity was increased to 150 mM NaCl in the presence (10 mM) or limitation (1 mM) of ammonium as a nitrogen source. Upon salinity, “100/1B” was shown to support N assimilation by enhancing the glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) cycle under high N, and the stimulation of the glutamate dehydrogenase (GDH) pathway under low N treatment. In “Barley medenine”, salinity reduced the GS/GOGAT cycle, and increased GDH activity. Upon salinity, Heat Shock Proteins 70 and PEPC remained unchanged in “100/1B”, while they decreased in “Barley medenine”. The tolerance degree is a determining factor in enzymes’ occurrence and regulation: exposed to salinity, “100/1B” rapidly increased APX and PEPC activities, while this was delayed in “Barley medenine”. Salinity increased cyt-G6PDH levels in “100/1B”, while “Barley medenine” showed a decrease in G6PDH isoforms. Correlation analyses confirm GOGAT was related to G6PDH; GDH and APX with PEPC in “100/1B” under moderate salinity; severe salinity correlated GDH with G6PDH and PEPC. In “Barley medenine” under salinity, GOGAT was correlated with G6PDH, while APX showed a relation with PEPC. Therefore, specific enzymatic activities and occurrence can be used to determine stress responsiveness of different landraces. We suggest that the rapid increase in G6PDH, APX, and nitrogen assimilation enzymes activities represents an index of tolerance in “100/1B” and a stress symptom in “Barley medenine”.

Rescue of myocardial energetic dysfunction in diabetes through the correction of mitochondrial hyperacetylation by honokiol.

Cardiac energetic dysfunction has been reported in patients with type 2 diabetes (T2D) and is an independent predictor of mortality. Identification of the mechanisms driving mitochondrial dysfunction, and therapeutic strategies to rescue these modifications, will improve myocardial energetics in T2D. We demonstrate using 31P-magnetic resonance spectroscopy (31P-MRS) that decreased cardiac ATP and phosphocreatine (PCr) concentrations occurred before contractile dysfunction or a reduction in PCr/ATP ratio in T2D. Real-time mitochondrial ATP synthesis rates and state 3 respiration rates were similarly depressed in T2D, implicating dysfunctional mitochondrial energy production. Driving this energetic dysfunction in T2D was an increase in mitochondrial protein acetylation, and increased ex vivo acetylation was shown to proportionally decrease mitochondrial respiration rates. Treating T2D rats in vivo with the mitochondrial deacetylase SIRT3 activator honokiol reversed the hyperacetylation of mitochondrial proteins and restored mitochondrial respiration rates to control levels. Using 13C-hyperpolarized MRS, respiration with different substrates, and enzyme assays, we localized this improvement to increased glutamate dehydrogenase activity. Finally, honokiol treatment increased ATP and PCr concentrations and increased total ATP synthesis flux in the T2D heart. In conclusion, hyperacetylation drives energetic dysfunction in T2D, and reversing acetylation with the SIRT3 activator honokiol rescued myocardial and mitochondrial energetics in T2D.

Priming of tomato seedlings with 2-oxoglutarate induces arsenic toxicity alleviatory responses by involving endogenous nitric oxide.

Metal toxicity in crop plants is a matter of scientific concern. Therefore, in recent years efforts have been made to minimize metal toxicity in crop plants. Out of various strategies, priming of seedlings with certain chemicals, like e.g. donors of signaling molecules, nutrients, metabolites or plant hormones has shown encouraging results. However, mechanisms related with the priming-induced mitigation of metal toxicity are still poorly known. Hence, we have tested the potential of 2-oxoglutarate (2-OG) priming in enhancing the arsenate (AsV ) toxicity tolerance in tomato seedlings along with deciphering the probable role of nitric oxide (NO) in accomplishing this task. Arsenate decreased growth, endogenous NO and nitric oxide synthase-like activity but enhanced the accumulation of As, which collectively led to root cell death. Arsenate toxicity also decreased some photosynthetic characteristics (i.e. Fv /Fm, qP, Fv /F0 and Fm /F0 , and total chlorophyll content) but enhanced NPQ. However, priming with 2-OG alleviated the toxic effect of AsV on growth, endogenous NO, cell death and photosynthesis. Moreover, arsenate inhibited the activities of enzymes of nitrogen metabolism (i.e. nitrate reductase, nitrite reductase, glutamine synthetase and glutamine 2-oxoglutarate aminotransferase) but increased the activity of glutamate dehydrogenase and NH4 + content. Superoxide radicals, hydrogen peroxide, lipid peroxidation, protein oxidation and membrane damage increased upon AsV exposure, but the antioxidant enzymes (i.e. superoxide dismutase, catalase and glutathione-S-transferase) showed differential responses. Overall, our results showed that 2-OG is capable of alleviating AsV toxicity in tomato seedlings but the involvement of endogenous NO is probably required.

Assimilation of exogenous cyanide cross talk in Oryza sativa L. to the key nodes in nitrogen metabolism.

Exogenous cyanide (CN-) effects on nitrogen (N) uptake, transport, and assimilation in rice seedlings were investigated at the biochemical and molecular levels. Seedlings were treated with either a 2-d or 4-d supply of potassium cyanide (KCN) in the nutrient solution containing nitrate (NO3-) or ammonium (NH4+). Although a KCN-induced increase was recorded in the activity of β-cyanoalanine synthase (β-CAS) in rice tissues of both NH4+-fed and NO3--fed seedlings, the former showed a significantly greater assimilation rate for CN- than the latter. The addition of KCN decreased NO3- uptake and assimilation, whereas a negligible impact was observed in NH4+ treatments. Enzymatic assays showed a marked activities enhancement of glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in NH4+-fed seedlings after KCN exposure. Similarly, the rises occurring in nitrate reductase (NR) and GDH activity in NO3--fed seedlings were also detected after 2-d exposure to KCN, whereas a significant reduction of GS and GOGAT activities was determined. The results suggest that although exogenous KCN at moderate or high concentrations caused repressively effects on biomass growth of both NH4+-fed and NO3--fed rice seedlings, the nontoxic concentration of KCN supplied can serve as a supplemental N source in plant nutrition and N metabolism.

Quercetin attenuates endocrine and metabolic responses to oxytetracycline in silver catfish (Rhamdia quelen)

This study aimed to verify whether dietary quercetin protects against the detrimental effects induced by oxytetracycline (OTC) administration in silver catfish (Rhamdia quelen). Fish were divided into different experimental groups that received OTC and/or quercetin, either during 14 or 21 days. To determine the endocrine system stress response, we have measured the brain mRNA expression levels of corticotropin-releasing hormone (crh), proopiomelanocortins (pomca and pomcb) and some of the pituitary hormones (growth hormone [gh], somatolactin [sl], and prolactin [prl]). We have also quantified the levels of cortisol as well as some metabolites (glucose, glycogen, lactate, and triglycerides) in the plasma. Moreover, the enzymatic activity of hexokinase, phosphorylase (active GPase), fructose-biphosphatase (FBP), glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate dehydrogenase (GDH) and gill Na+/K+-ATPase were measured. The results demonstrated that OTC activates the silver catfish stress response by increasing the plasma cortisol and decreasing the glucose levels at 14 and 21 days. Additionally, OTC also altered the fish hepatic metabolic status as demonstrated by an increase in triglycerides levels and the enzymatic activity of both FBP and GDH after 14 days. OTC also stimulated Na+/K+-ATPase activity in the gill after 14 days and altered the hypophyseal expression of gh (at 14 and 21 days) and prl (at 14 days). The co-treatment with 1.5 g of quercetin could prevent most of the alterations caused by OTC, strongly suggesting quercetin as a beneficial compound when added to the fish diet.

Valine acts as a nutritional signal in brain to activate TORC1 and attenuate postprandial ammonia-N excretion in Chinese perch (Siniperca chuatsi).

An emerging concept is that the hypothalamic nutrient sensor can regulate peripheral energy metabolism via a brain-liver circuit. Valine is an essential branched-chain amino acid (BCAA) that drives intracellular signaling cascades by the activation of target of rapamycin complex 1 (TORC1) which is critical to protein metabolism in mammals. However, in teleost fish, it remains scarce in this area especially about how the intraventricular (ICV) injection of valine can mediate the protein metabolism in peripheral organs. This study would tentatively explore the effects of ICV injection of valine on protein metabolism in peripheral organs through evaluating the postprandial ammonia-N excretion rate in Chinese perch. The control group was injected with 5-μL PBS, and the Val group was injected with 20-μgL valine dissolved into 5-μL PBS. The ammonia-N excretion rate of Val group was lower than control group at 4-, 12-, and 24-h postinjection, while the concertation of plasma glucose was increased sharply at 0.5-, 4-, 12-, and 24-h postinjection. We further checked both mRNA level and the enzyme activity of glutamate dehydrogenase (GDH) in the liver and adenosine monophosphate deaminase (AMPD) in muscle, and we found that they were obviously decreased in Val group at 4-, 12-, and 24-h postinjection. The phosphorylation level of ribosomal protein S6, a downstream target protein of TORC1, was markedly enhanced in the liver of Val group at 4-, 12-, and 24-h postinjection. Collectively, these results illustrated that ICV injection of valine can attenuate protein degradation in peripheral organs by depressing the GDH and AMPD enzyme activity; on the other hand, the injected valine can trigger the activation of TORC1 in the liver via a brain-liver circuit and then interdict proteolysis.

Overactivation of glutamate consuming pathways in l-glutamate treated tomato fruits lead to resistance against Alternaria Alternata

Abstract Previous studies reported the ability of l -glutamate (glutamate) to induce host resistance against Alternaria alternata, but the underlying mechanism is still scarce. Here, temporal alteration in antioxidant enzymes and glutamate consuming pathways were transcriptionally and enzymatically monitored in tomato fruits. The results showed that exogenous glutamate application enhanced the activities of antioxidant enzymes, including peroxidase, superoxide dismutase and catalase, in tomatoes upon inoculation with Alternaria alternata, while lower concentrations of H2O2 and malondialdehyde were observed. Interestingly, glutamate led to a decline in carbon:nitrogen ratio and induction of deaminating activity of glutamate dehydrogenase, resulting in replenishment of the tricarboxylic acid cycle. Concurrently, proline dehydrogenase mediated the proline catabolism was downregulated by glutamate, accompanied by the enhanced biosynthesis of proline due to increased expression of pyrroline-5-carboxylate synthase. Together our findings favored a model whereby overactivation of antioxidant enzymes and glutamate consuming pathways, deamination of glutamate and proline biosynthesis from glutamate, played a vital role in the mechanism of glutamate-induced resistance.

Effects of nitric oxide on nitrogen metabolism and the salt resistance of rice (Oryza sativa L.) seedlings with different salt tolerances

Salt stress inhibits rice productivity seriously. Nitric oxide (NO) is an endogenous signaling molecule in plants that can improve the resistance of rice to abiotic stresses. Previous studies also showed that nitrogen metabolism is essential for rice stress-tolerance. However, the physiological and molecular mechanisms by how NO affects the nitrogen metabolisms of rice seedlings remain unclear. A hydroponic experiment with two rice varieties, Jinyuan85 (salt tolerant) and Liaojing763 (salt sensitive), was carried out to explore whether NO could alleviate the negative effects of salt stress on nitrogen metabolism and increase salt resistance of rice seedlings. The results showed that (1) the application of NO alleviated the inhibitory effects of salt stress on plant height and biomass accumulation, and increased the nitrogen content of rice leaf. (2) the accumulation of the sucrose and proline was markedly increased in salt stress after application of NO, and peroxidase activities was increased by 107% and 67.7% for Jinyuan85 and Liaojing763, respectively. (3) NO significantly increased the activities of glutamate dehydrogenase, sucrose synthase and sucrose phosphate synthase in both rice varieties under salt stress. (4) Additionally, NO regulated the expression levels of AMT, NIA and SUT genes, but these regulation effects are different with rice varieties and treatments. The results suggested that NO mainly increased the glutamate dehydrogenase and peroxidase activities and sucrose accumulation to enhance the nitrogen metabolism and antioxidative capacity, and alleviated the negative effects of salt stress on rice performance.

Potential of Seed Halopriming in Mitigating NaCl-induced Adversities on Nitrogen Metabolism in Legume Crops

Background: Salinity is a major threat that impairs legume growth and development worldwide. Therefore, present study was aimed to determine the potential of seed halopriming in relieving NaCl-induced disturbances on nitrogen metabolism of seedlings of six legume crops viz., Lens culinaris, Cajanus cajan, Cicer arietinum, Lathyrus sativus, Vigna radiata and Vigna mungo that were detected to have differential sensitivity to NaCl. Methods: Nonprimed and haloprimed seeds were grown hydroponically under varying NaCl doses for three weeks. Harvested samples were utilised to characterize the toxic effects of NaCl on nitrogen metabolism of nonprimed and haloprimed seedlings. Results: Nonprimed seedlings exhibited reduced nitrate uptake by virtue of which other assimilatory processes of nitrogen fixation were adversely affected. Haloprimed seedlings experienced lesser toxicity under NaCl stress due to elevated activities of nitrate assimilatory enzymes on account of improved nitrate uptake from solution. Lesser ammonium accumulation and lower glutamate dehydrogenase activity implied lesser cytotoxicity in primed seedlings. Based on the trends obtained from tested parameters, nitrogen metabolism was maximally affected in Lens and Cajanus followed by Cicer and Lathyrus. Vigna radiata and Vigna mungo were least affected and therefore may be suggested for cultivation in saline prone agricultural fields after seed halopriming.

Oxidation of energy substrates in tissues of largemouth bass (Micropterus salmoides).

This study tested the hypothesis that amino acids are oxidized at higher rates than glucose and palmitate for ATP production in tissues of largemouth bass (LMB, a carnivorous fish). Slices (10 to 50 mg) of liver, proximal intestine, kidney, and skeletal muscle isolated from LMB were incubated at 26°C for 2h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5mM D-glucose) containing either D-[U-14C]glucose, 2mM L-alanine plus L-[U-14C]alanine, 2mM L-aspartate plus L-[U-14C]aspartate, 2mM L-glutamate plus L-[U-14C]glutamate, 2mM L-glutamine plus L-[U-14C]glutamine, 2mM L-leucine plus L-[U-14C]leucine, or 2mM palmitate plus [U-14C]palmitate. In parallel experiments, tissues were incubated with a [U-14C]-labeled tracer and a mixture of unlabeled substrates [alanine, aspartate, glutamate, glutamine, leucine, and palmitate (2mM each) plus 5mM glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In separate experiments, O2 consumption by each tissue was measured in the presence of individual or a mixture of substrates. The activities of key metabolic enzymes were also measured. Results indicated that the liver and skeletal muscle had a limited ability to oxidize glucose and palmitate to CO2 for ATP production in the presence of individual or a mixture of substrates due to low activities of carnitine palmitoyltransferase-I, hexokinase and pyruvate dehydrogenase. In the presence of individual substrates, each amino acid was actively oxidized by all the tissues. In the presence of a mixture of substrates, glutamine and glutamate were the major metabolic fuels in the proximal intestine and kidney, as glutamine for the liver and aspartate for skeletal muscle. All the tissues had high activities of glutaminase, glutamate dehydrogenase, and transaminases. At the same extracellular concentration of amino acids (2 mM) in a mixture of energy substrates, glutamine was the major metabolic fuel for the liver of the LMB, glutamine and glutamate for the proximal intestine and kidneys, and aspartate for the skeletal muscle. Glutamine plus glutamate plus aspartate generated 60-70% of ATP in LMB tissues.

Quantitative proteomics analysis of tomato growth inhibition by ammonium nitrogen

As a single nitrogen source, ammonium (NH4+) can inhibit the growth of plants, especially when applied in excess. Tandem mass tag (TMT) quantitative proteomics technology was employed in the current study to explore and analyze the mechanisms of ammonium-induced inhibition. F1 tomato (Lycopersicon esculentum Mill) was used in this study. Seedlings at the four leaf-stages grown in a greenhouse were irrigated using nutrient solution with NH4+-N as single nitrogen source (15 mmol L−1, single NO3−-N as control) for 5 weeks. Compared to the control, the root biomass of NH4+-N-treated seedlings decreased by 50%. In addition, NH4+ content in roots was 2.83-fold increased and soluble sugar and protein contents were increased. However, the starch content did not change significantly. The activities of glutamine synthetase (GS), glutamate synthetase (GOGAT) and glutamate dehydrogenase (GDH), which are involved in ammonium assimilation, were increased, and glutamine (Gln) content was also increased. However, glutamate (Glu) content, which is important for amino transfer, did not significantly increase. Ammonium assimilation was inhibited. Root quantitative proteomics showed that carbonic anhydrase Q5NE21 was significantly downregulated. Although K4BPV5 and K4D9J3 proteins, which improve ammonium assimilation, were upregulated, ammonium assimilation was limited. In addition, NH4+ accumulated, which is likely due to Q5NE21 downregulation. Meanwhile, cell wall metabolism related to phenylpropanoid biosynthesis was altered due to the accumulation of NH4+ levels. Subsequently, tomato root growth was inhibited.

Diclofenac shifts the role of root glutamine synthetase and glutamate dehydrogenase for maintaining nitrogen assimilation and proline production at the expense of shoot carbon reserves in Solanum lycopersicum L.

The continuous increase of the human population worldwide has led to an increase of pharmaceuticals' consumption, such as diclofenac (DCF), a widely used non-steroidal anti-inflammatory drug (NSAID), that is not removed by wastewater treatment processes. Although there is some research regarding the effects of DCF on animals and aquatic invertebrates, information concerning its influence on plants' metabolism is still scarce. Through an integrated approach, using combined biochemical and molecular biology techniques, this work aimed to evaluate the phytotoxicity of DCF in Solanum lycopersicum L., focusing on the primary plant processes: nitrogen (N) assimilation and photosynthesis. The exposure of tomato plants to increasing concentrations of DCF (0, 0.5, and 5 mg L-1) revealed that glutamine synthetase (GS) was differentially affected, in an organ-dependent manner, by this contaminant at the gene expression, protein, and activity levels, with an increased activity of 0.2-fold in shoots of plants treated with the lowest concentration of DCF although a general decrease was registered for the SlGS gene family expression, revealing that post-translational regulation was in order, since GS2 polypeptide content did not change. Glutamate dehydrogenase (GDH) activity was generally enhanced, accompanied by increases of 0.4- to 1.9-fold in proline levels, revealing GDH as an important compensatory route for both N assimilation and proline production under stressful conditions. No alterations in most photosynthetic endpoints were noticed after DCF treatments, but small decreases of 0.1- to 0.8-fold in the accumulation of RuBisCO-encoding transcripts were observed, along with a reduction in starch content. Some alterations in the soluble polypeptide profile were also detected in response to DCF, evidencing the participation of some stress-related proteins in the plant's response to DCF.

Serum glutamate dehydrogenase activity enables early detection of liver injury in subjects with underlying muscle impairments

Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.

Effect of a mist culture system on photosynthesis and nitrogen metabolism in ginger.

To evaluate the transition from traditional shading cultivation to mist cultivation, a field experiment was carried out. The results demonstrated that compared with traditional shading, the mist treatment significantly reduced leaf temperature. Likewise, the higher transpiration rate also contributes to reducing leaf temperature and protects ginger from heat stress in summer. Moreover, a higher instantaneous efficiency of water use suggested that water lost via transpiration was beneficial under a mist culture system. The higher instantaneous efficiency of water use in the mist treatment was caused mainly by the higher net photosynthetic rate, which is further reflected by the higher rhizome yield of ginger under the mist culture system. Instead of lowering the temperature by lowering photon flux density, mist treatment does not seriously reduce the photon flux density while reducing the temperature of the blade. Hence, the net photosynthetic rate in the shading treatment is significantly lower than that in the mist treatment, although the maximal quantum yield of photosystem II and the actual photochemical efficiency of photosystem II in ginger in the shading treatment were significantly higher than those in the mist treatment. Lower superoxide anion, hydrogen peroxide, and malondialdehyde contents were also found after mist treatment. Lower ammonium avoids the potential risk of ammonium toxicity and is based on higher nitrate reductase, glutamine synthetase, and glutamate synthase activity but lower glutamate dehydrogenase activity. Therefore, the mist cultivation system improved the physiological characteristics and yields of ginger and can be suggested as an alternative, sustainable, and cleaner cultivation measure.