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[EN] Twenty lactic acid bacteria (LAB) isolated from artisanal cheeses and previously selected for their technological properties were screened for their enzymatic activities, antimicrobial activity and safety. The aim was to select those LAB strains that were safe and showed advantageous properties for the development of starter cultures for cheese making, discarding those that could transfer antibiotic resistance or produce any toxic biogenic amines. Aminopeptidase activities were detected in most strains, particularly high for the substrate leucine arylamidase, and most lactobacilli and Leuconostoc strains showed high β-galactosidase activity. Glutamate dehydrogenase (GDH) activity was detected in 13 strains, although the activity values varied widely. All strains showed antimicrobial activity against the indicator microorganisms due to acid production. However, only one of the Lactiplantibacillus plantarum strains showed an inhibitory activity against Enterococcus faecalis due to bacteriocin-like compounds. In particular, Levilactobacillus brevis TAUL1567 and Lactiplantibacillus paraplantarum TAUL1399 showed resistance to tetracycline and ampicillin, respectively, above the cut-off values and were therefore excluded. Tyramine was only produced by L. brevis TAUL1567 (193.15 μg ml−1), while putrescine was produced by this strain and two strains of Lactococcus lactis. Finally, 14 strains produced γ-aminobutyric acid (GABA), five of them at concentrations around or above 100 μg ml−1. SI

Carbon nanodots modified with azure A (AA-CDs) have been synthesized and applied as redox mediator of bioelectrocatalytic reactions. A deep characterization of AA-CDs nanomaterial has been carried out, proving the covalent attachment of azure A molecules into the carbon dots nanostructure. Disposable screen-printed carbon electrodes (SPCE) have been modified with AA-CDs, through the action of chitosan polymer (Chit-AA-CDs/SPCE). The Chit-AA-CDs/SPCE electrocatalytic activity towards the oxidation of NADH has been proved, obtaining excellent results regarding the low oxidation potential achieved (−0.15 V vs. Ag) and low detection and quantification limits (LOD and LOQ) for NADH, 16 and 53 µM, respectively. The developed electrochemical platform has been applied for the construction of a glutamate biosensor by immobilizing L-glutamic dehydrogenase (GLDH/Chit-AA-CDs/SPCE). The morphology of GLDH/Chit-AA-CDs/SPCE platform was analysed by AFM at each different step of the electrode modification process. The resulting biosensing platform is capable of detect NADH enzymatically generated by GLDH in the presence of glutamate and NAD+. Good analytical parameters were obtained for glutamate analysis using GLDH/Chit-AA-CDs/SPCE, as LOD and LOQ of 3.3 and 11 µM, respectively. The biosensor has been successfully applied to the analysis of food and biological samples, This work has been supported by the Spanish Ministerio de Ciencia e Innovacion (PID2020–116728RB-I00) and Comunidad Autonoma de Madrid (SI3/PJI/2021–00341, P2018/NMT-4349 TRANSNANOAVANSENS Program)

Chromium (Cr) is a dangerous metal that has been found in the environment in high amounts as the result of numerous human activities, with proven negative effects on various organisms, including plants. The exposure of some plant species to this metal resulted in reduced growth, impaired nutrient uptake, and led to an overproduction of reactive oxygen species (ROS), ultimately causing oxidative damage. Nevertheless, studies regarding the impact of Cr on nitrogen (N) metabolism, the main limiting factor for plant growth, either directly or through the occurrence of oxidative stress, are scarce. Thus, this work aimed to explore the interplay between ammonia (NH4+) assimilation, redox homeostasis and antioxidant (AOX) system of tomato plants exposed to increased concentrations of Cr (VI) (0, 5 and 10 µM). The results revealed that Cr (VI) induced biometric damages, with roots being the most affected organ. Glutamine synthetase (GS) was differentially influenced by the metal at the gene expression, protein and activity levels, whereas glutamate dehydrogenase (GDH) activity was enhanced. Moreover, in Cr (VI)-treated plants, the observed oxidative damage was dependent on metal concentration and plant organ, with a differential accumulation of ROS and a generalized increase of lipid peroxidation (LP) recorded, being paired with an overall failure of the enzymatic antioxidant (AOX) system. Still, the non-enzymatic AOX component had a relevant role in the protection of plants against Cr (VI). Overall, the obtained results suggest that Cr (VI)-mediated plant growth reduction resulted from the occurrence of oxidative stress, coupled with the lack of enzymatic AOX defense, which, consequently, affected the NH4+ assimilatory route.

Glutamate and dopamine hypotheses are leading theories of the pathophysiology of schizophrenia. Multiple lines of evidence suggest that dopaminergic and glutamatergic dysfunction is an underlying mechanism in schizophrenia. Since currently available antipsychotic drugs have significant untoward side effects, identification of new neuroprotective compounds from the medicinal plants may prove beneficial in neurodegenerative disorders. In our previous investigation we have isolated, characterized and reported a novel bioactive compound viz. 3-(3, 4-dimethoxy phenyl)-1-(4-methoxy phenyl) prop-2-en-1-one from the Celastrus paniculatus (CP) is used for the current clinical intervention of schizophrenia disease. The present study is mainly aimed to evaluate the neuroprotective potential of the above bioactive compound against ketamine-induced schizophrenia with particular reference to glutamate metabolism using in vivo and in silico methods. The decrease in glutamine content and the activity levels of glutamate dehydrogenase, glutamine synthetase, and glutaminase in different regions of the rat brain suggests lowered oxidative deamination and lowered mobilization of glutamate towards glutamine formation during ketamine-induced schizophrenia. Pre-treatment with the plant compound reversed the alterations in glutamate metabolism and restored the normal glutamatergic neurotransmission akin to the reference drug, clozapine. In addition, the compound has shown strong interaction and exhibited the highest binding energies against selected NMDA receptors with the lowest inhibition constant than the reference drug. Recoveries of these parameters during anti-schizophrenic treatment suggest that administration of plant compound might offer neuroprotection by interrupting the pathological cascade of glutamatergic neurotransmission that occurs during schizophrenia.

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., This work was funded by Spanish Ministry of Science and Innovation - MICINN (AGL2016-76069-C2-1-R) awarded to JMM. IJC was supported by a contract from the University of Cádiz (PIF UCA/REC02VIT/2014; 2018-011/PU/AY.PUENTE/CD). The authors (IJC and JMM) belong to the Fish Welfare and Stress Network (AGL2016-81808-REDT), which is supported by the Agencia Estatal de Investigación (MICINN, Spanish Government). The authors wish to thank the financial support received by Conselho Nacional de Pesquisa e Desenvolvimento Científico-CNPq (Ministry of Education of Brazil, Brazil) through the program CSF (Programa Ciência sem Fronteiras (CONCF) - Process: 207329/2015-0) awarded to Tanise da Silva Pês and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

L-theanine is the most abundant free amino acid in tea that offers various favorable physiological and pharmacological effects. Bacterial enzyme of γ-glutamylmethylamide synthetase (GMAS) can catalyze the synthesis of theanine from glutamate, ethylamine and ATP, but the manufacturing cost is uncompetitive due to the expensive substrates and complex processes. In this study, we described pathway engineering of wild-type Escherichia coli for one-step fermentative production of theanine from sugars and ethylamine. First, the synthetic pathway of theanine was conducted by heterologous introduction of a novel GMAS from Paracoccus aminovorans. A xylose-induced T7 RNA polymerase-PT7 promoter system was used to enhance and control gmas gene expression. Next, the precursor glutamate pool was increased by overexpression of native citrate synthase and introduction of glutamate dehydrogenase from Corynebacterium glutamicum. Then, in order to push more carbon flux towards theanine synthesis, the tricarboxylic acid cycle was interrupted and pyruvate carboxylase from C. glutamicum was introduced as a bypath supplying oxaloacetate from pyruvate. Finally, an energy-conserving phosphoenolpyruvate carboxykinase from Mannheimia succiniciproducens was introduced to increase ATP yield for theanine synthesis. After optimizing the addition time and concentration of ethylamine hydrochloride in the fed-batch fermentation, the recombinant strain TH11 produced 70.6 ​g/L theanine in a 5-L bioreactor with a yield and productivity of 0.42 ​g/g glucose and 2.72 ​g/L/h, respectively. To our knowledge, this is the first report regarding the pathway engineering of E. coli for fermentative production of theanine. The high production capacity of recombinant strain, combined with the easy processes, will hold attractive industrial application potential for the future., Highlights • γ-Glutamylmethylamide synthetase from P. aminovorans showed high ligation activity. • Xylose-induced T7 RNA polymerase-PT7 promoter system was used to control gene expression. • TCA cycle was rewired to push more carbon flux toward theanine synthesis. • Ethylamine feeding strategy was optimized to balance cell growth and theanine production.

Giardia duodenalis is one of the most common causes of diarrhea in humans with about 250–300 million cases per year. It is considered to be a species complex comprising of eight genetic assemblages (A to H), with assemblages A and B being the major causes of human infections. In this study we carried out genotypic characterization of G. duodenalis isolates detected in asymptomatic school-going children aged 3–16 years. Between May and September 2017, a total of 329 fecal samples were collected from school-going children from Chawama compound of Lusaka City and were screened for Giardia by microscopic examination. All microscopically positive fecal samples were analyzed by semi-nested polymerase chain reaction (PCR) targeting the glutamate dehydrogenase (gdh) gene. Genotyping of amplified PCR products was conducted by restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Microscopically, Giardia was found in 10% (33/329) of fecal samples. The PCR-RFLP analysis of the gdh gene revealed assemblages A and B in 27.3% (9/33) and 72.7% (24/33), respectively. Furthermore, analysis with restriction enzymes identified sub-assemblages AII (27.3%, 9/33), BIII (12.1%, 4/33), BIV (51.5%, 17/33) and mixed infections of BIII and BIV (9.1%, 3/33). Phylogenetic analysis showed the clustering of 27.6% (8/29) and 72.4% (21/29) of Zambian Giardia gdh gene sequences into assemblages A and B, respectively. This study has revealed the presence of both assemblage A and B and that spread of G. duodenalis in school-going children appears to be mostly through anthroponotic transmission. To our knowledge, this is the first report of genotypic characterization of G. duodenalis identified in Zambia.

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates.

Cancer cells rapidly consume glutamine as a carbon and nitrogen source to support proliferation, but the cell number continues to increase exponentially after glutamine is nearly depleted from the medium. In contrast, cell proliferation rates are strongly depressed when cells are cultured in glutamine-free medium. How cancer cells survive in response to nutrient limitation and cellular stress remains poorly understood. In addition, rapid glutamine catabolism yields ammonia, which is a potentially toxic metabolite that is secreted into the extracellular space. Here, we show that ammonia can be utilized for glutamate production, leading to cell proliferation under glutamine-depleted conditions. This proliferation requires glutamate dehydrogenase 2, which synthesizes glutamate from ammonia and cc-ketoglutarate and is expressed in MCF7 and T47D cells. Our findings provide insight into how cancer cells survive under glutamine deprivation conditions and thus contribute to elucidating the mechanisms of tumor growth.

Clostridium difficile has not been studied in detail in Asia, particularly Southeast Asia. We thus performed a prevalence study across four hospitals in Central Java province, Indonesia. Stool samples were collected from patients with diarrhoea and tested by enzyme immunoassay for glutamate dehydrogenase (GDH) and toxin A/B (C DIFF QUIK CHEK COMPLETE, TechLab). Specimens were cultured and molecular typing was performed. In total, 340 samples were tested, of which 70 (20.6%) were GDH positive, with toxin detected in 19 (5.6%). Toxigenic C. difficile was isolated from 37 specimens (10.9%), while a further 36 (10.6%) nontoxigenic isolates were identified. The most common strain was ribotype 017 (24.3% of 74 isolates), followed by nontoxigenic types QX 224 (9.5%), and QX 238 and QX 108 (both 8.1%). The high prevalence of C. difficile highlights a need for ongoing surveillance of C. difficile infection in Indonesia.

Development of the new crop cultivars with high yield under low nitrogen (N) input is a fundamental approach to enhance agricultural sustainability, which is dependent on the exploitation of the elite germplasm. In the present study, four barley genotypes (two Tibetan wild and two cultivated), differing in N use efficiency (NUE), were characterized for their physiological and biochemical responses to different N levels. Higher N levels significantly increased the contents of other essential nutrients (P, K, Ca, Fe, Cu and Mn), and the increase was more obvious for the N-efficient genotypes (ZD9 and XZ149). The observation of ultrastructure showed that chloroplast structure was severely damaged under low nitrogen, and the two high N efficient genotypes were relatively less affected. The activities of the five N metabolism related enzymes, i.e., nitrate reductase (NR), glutamine synthetase (GS), nitrite reductase (NiR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) all showed the substantial increase with the increased N level in the culture medium. However the increased extent differed among the four genotypes, with the two N efficient genotypes showing more increase in comparison with the other two genotypes with relative N inefficiency (HXRL and XZ56). The current findings showed that a huge difference exists in low N tolerance among barley genotypes, and improvement of some physiological traits (such as enzymes) could be helpful for increasing N utilization efficiency.

11 pages, 8 figures, 2 tables, Anisakis simplex is a parasitic nematode that can cause anisakiosis and/or allergic reactions in humans. The presence of invasive third-stage larvae (L3) in many different consumed fish species and the fourth-stage larvae (L4) in marine mammals, where L3 can accidentally affect to humans and develop as far as stage L4. World Health Organization and food safety authorities aim to control and prevent this emerging health problem. In the present work, using Tandem Mass Tag (TMT)-based quantitative proteomics we analyzed for the first time the global proteome of two A. simplex development stages, L3 and L4. The strategy was divided into four steps: (a) protein extraction of L3 and L4 development stages, (b) high intensity focused ultrasound (HIFU)-assisted trypsin digestion, (c) TMT-isobaric mass tag labeling following by high-pH reversed-phase fractionation, and (d) LC-MS/MS analysis in a LTQ-Orbitrap Elite mass spectrometer. A total of 2443 different proteins of A. simplex were identified. Analysis of the modulated proteins provided the specific proteomic signature of L3 (i.e. pseudocoelomic globin, endochitinase 1, paramyosin) and L4 (i.e. neprilysin-2, glutamate dehydrogenase, aminopeptidase N). To our knowledge, this is the most comprehensive dataset of proteins of A. simplex for two development stages (L3 and L4) identified to date, This work was supported by the Ramón Areces Foundation (XVII National grant), the GAIN-Xunta the Galicia Project (IN607D 2017/01), and as part of the project “Development Program of the University of Warmia and Mazury in Olsztyn” no. POWR.03.05.00-00-Z310/17

The liver is particularly vulnerable to chemical-induced injury, owing to its central role in maintaining metabolic homeostasis and its location between the digestive tract and the rest of the body. Because venous blood from the stomach and intestines enters the liver via the portal vein prior to entering systemic circulation, the liver is the first organ to encounter ingested nutrients, vitamins, metals, drugs, and environmental chemicals, including waste products from intestinal bacteria. The first-pass exposure to drugs administered orally and the high capacity for xenobiotic metabolism are considered significant reasons why the liver is a target for drug toxicity, and this aspect has been widely reviewed (Park et al., 2005). Toxic metabolites can be generated and accumulate within hepatocytes as a result of oxidation–reduction (phase I), conjugation (phase II), and the saturation of transporter (phase III) reactions which normally serve to remove or detoxify xenobiotics (Park et al., 2005, 2011). Acute or chronic exposure to chemicals can detrimentally alter all or some of the key functions of the liver, including the scavenging or uptake processes that extract absorbed materials from the blood and process them via catabolism, storage, and/or biliary excretion. Such disruption can occur with or without appreciable cellular damage (Treinen-Moslen, 2001). As such, detection of chemically induced liver injury can be complicated and is largely reliant upon tests that measure integrity of liver cells as well as hepatic function. The primary purpose of this article is to discuss both established and emerging tests for the detection of hepatotoxicity in mammalian test species and in clinical practice.

The GluD1 and GluD2 receptors form the GluD ionotropic glutamate receptor (iGluR) subfamily. Without known endogenous ligands, they have long been referred to as 'orphan' and remained enigmatic functionally. Recent progress has, however, radically changed this view. Both GluD receptors are expressed in wider brain regions than originally thought. Human genetic studies and analyses of knockout mice have revealed their involvement in multiple neurodevelopmental and psychiatric disorders. The discovery of endogenous ligands, together with structural investigations, has opened the way towards a mechanistic understanding of GluD signaling at central nervous system synapses. These studies have also prompted the hypothesis that all iGluRs, and potentially other neurotransmitter receptors, rely on the cooperative binding of extracellular small-molecule and protein ligands for physiological signaling.

[Background] Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide +–dependent histone deacetylase that regulates various pathways involved in ischemia-reperfusion injury (IRI). Moreover, high-mobility group box 1 protein (HMGB1) has also been involved in inflammatory processes during IRI. However, the roles of both SIRT1 and HMGB1 in liver preservation is poorly understood. In this communication, we evaluated the potential relationship between SIRT1 and HMGB1 in steatotic and non-steatotic liver grafts preserved in Institute Georges Lopez solution (IGL-1) preservation solution enriched or not enriched with trimetazidine (TMZ)., [Methods] Steatotic and non-steatotic livers were preserved in IGL-1 preservation solution (24 hours, 4°C), enriched or not enriched with TMZ (10 μmol/L), and then submitted to ex vivo reperfusion (2 hours; 37°C). Liver injury (AST/ALT) and function (bile output, vascular resistance) were evaluated. SIRT1, HMGB1, autophagy parameters (beclin-1, LC3B), PPAR-γ, and heat-shock protein (HO-1, HSP70) expression were determined by means of Western blot. Also, we assessed oxidative stress, mitochondrial damage (glutamate dehydrogenase), and TNF-α levels., [Results] Elevated SIRT1 and enhanced autophagy were found after reperfusion in steatotic livers preserved in IGL-1+TMZ when compared with IGL-1. However, these changes were not seen in the case of non-steatotic livers. Also, HO-1 increases in the IGL-1 + TMZ group were evident only in the case of steatotic livers, whereas HSP70 and PPAR-γ protein expression were enhanced only in non-steatotic livers. All reported changes were consistent with decreased liver injury diminution, ameliorated hepatic function, and decreased TNF-α and HMGB levels. In addition, the oxidative stress and mitochondrial damage were efficiently prevented by the IGL-1 + TMZ use., [Conclusions] SIRT1 is associated with HMGB1 decreases and increased autophagy in steatotic livers, contributing to increased tolerance to cold IRI., This work was supported by Instituto de Salud Carlos III (ISCIII) through the FIS project PI12/0056, co-funded by FEDER from Regional Development European Funds (European Union).

Summary Objectives To evaluate the performance of the GeneXpert C. difficile assay and C. diff Quik Chek Complete (QCC) kit for the detection of toxins from fecal specimens and cooked meat broth (CMB) culture using toxigenic stool culture as reference method, for the diagnosis of C . difficile infection (CDI) in a community setting. Methods Non-repeat stool samples were tested simultaneously by GeneXpert and QCC. Toxin detection was done on neat stool samples, inoculated CMB, and isolated colonies. Results Nineteen (4.6%) of 409 samples were positive for glutamate dehydrogenase (GDH) in stool and CMB by the QCC assay; seven (1.7%) were positive for both GDH and toxins A/B. The sensitivities of QCC to detect C. difficile toxin directly in stool specimens and CMB were 68.4% and 100%, respectively, while specificities were 100% and 83%, respectively. C. difficile toxin was detected in 10 (2.5%) specimens and 13 (3.2%) CMB. Thirteen (68.4%) of 19 isolates were positive for C. difficile toxin by GeneXpert and QCC and were taken as the reference toxigenic culture. The disease burden was thus 3.2%. The sensitivities of GeneXpert in stool and CMB were 81.3% and 100%, respectively, while specificities were 100% and 100%, respectively. Conclusion The GeneXpert assay was more sensitive than QCC for the detection of C. difficile toxin in stool, but both assays were highly specific.

Ebselen is a synthetic, lipid-soluble seleno-organic compound. The high electrophilicity of ebselen enables it to react with multiple cysteine residues of various proteins. Despite extensive research on ebselen, its target molecules and mechanism of action remains less understood. We performed biochemical as well as in vivo experiments employing budding yeast as a model organism to understand the mode of action of ebselen. The growth curve analysis and FACS (florescence activated cell sorting) assays revealed that ebselen exerts growth inhibitory effects on yeast cells by causing a delay in cell cycle progression. We observed that ebselen exposure causes an increase in intracellular ROS levels and mitochondrial membrane potential, and that these effects were reversed by addition of antioxidants such as reduced glutathione (GSH) or N-acetyl-l-cysteine (NAC). Interestingly, a significant increase in ROS levels was noticed in gdh3-deleted cells compared to wild-type cells. Furthermore, we showed that ebselen inhibits GDH function by interacting with its cysteine residues, leading to the formation of inactive hexameric GDH. Two-dimensional gel electrophoresis revealed protein targets of ebselen including CPR1, the yeast homolog of Cyclophilin A. Additionally, ebselen treatment leads to the inhibition of yeast sporulation. These results indicate a novel direct connection between ebselen and redox homeostasis., Graphical Abstract, Highlights • Ebselen treatment causes an increase in intracellular ROS levels and mitochondrial membrane potential. • Ebselen inactivates GDH3 and Cyclophilin A by interacting with their cysteine residues. • The ebselen-induced increase in ROS levels leads to inhibition of sporulation in yeast. • Yeast gdh3 deletion potentiates ROS generation upon ebselen treatment.

The characterization of blood metabolite concentrations over the circadian period and across physiological stages is important for understanding the biological basis of feed efficiency, and may culminate in indirect methods for assessing feed efficiency. Hematological analyses for albumin, urea, creatine kinase, glutamate dehydrogenase, aspartate aminotransferase, carbon dioxide, and acetate were carried out in growing and gestating heifers. These measures were carried out in a sample of 36 Bos taurus crossed beef heifers held under the same husbandry conditions. Hourly blood samples were collected over a 24-h period on three separate sampling occasions, corresponding approximately to the yearling (and open), early-gestation and late-gestation stages. This design was used to determine variation throughout the day, effects due to physiological status and any associations with feed efficiency, as measured by residual feed intake. Blood analyte levels varied with time of day, with the most variation occurring between 0800 and 1600 h. There were also considerable differences in analyte levels across the three physiological stages; for example, creatine kinase was higher (P

Plant ammonium tolerance has been associated with the capacity to accumulate large amounts of ammonium in the root vacuoles, to maintain carbohydrate synthesis and especially with the capacity of maintaining high levels of inorganic nitrogen assimilation in the roots. The tricarboxylic acid cycle (TCA) is considered a cornerstone in nitrogen metabolism, since it provides carbon skeletons for nitrogen assimilation. The hypothesis of this work was that the induction of anaplerotic routes of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (NAD-ME) would enhance tolerance to ammonium nutrition. An experiment was established with tomato plants (Agora Hybrid F1) grown under different ammonium concentrations. Growth parameters, metabolite contents and enzymatic activities related to nitrogen and carbon metabolism were determined. Unlike other tomato cultivars, tomato Agora Hybrid F1 proved to be tolerant to ammonium nutrition. Ammonium was assimilated as a biochemical detoxification mechanism, thus leading to the accumulation of Gln and Asn as free amino acids in both leaves and roots as an innocuous and transitory store of nitrogen, in addition to protein synthesis. When the concentration of ammonium in the nutrient solution was high, the cyclic operation of the TCA cycle seemed to be interrupted and would operate in two interconnected branches to provide α-ketoglutarate for ammonium assimilation: one branch supported by malate accumulation and by the induction of anaplerotic PEPC and NAD-ME in roots and MDH in leaves, and the other branch supported by stored citrate in the precedent dark period. © 2013 Elsevier GmbH., This research was financially supported by the Basque Government (IT526-10) and by the Spanish Government (MICINN-AGL 2009-13339-CO2-01, AGL 2012-37815-C05-02). Technical support provided by G. Garijo from the Department of Natural Environmental Science (Universidad Pública de Navarra, UPNa) for amino acids measurements and by Dr. A. González (Phytotron Service, SGIker (Universidad del País Vasco, UPV/EHU) are gratefully acknowledged. We are also grateful to Dr. J.F Moran and Dr. K.A Roubelakis-Angelakis for lending GS and GDH antibodies, respectively.

Glutamate dehydrogenase (GDH) is found in all living organisms and catalyzes the reversible oxidative deamination of L-glutamate. Mammalian GDH is allosterically regulated by a wide array of metabolites. The importance of this allosteric regulation was made evident by the discovery of the hyperinsulinism/hyperammonemia (HHS) disorder. HHS is caused by spontaneous mutations in GDH that abrogate GTP inhibition. In the pancreas, this causes hypersecretion of insulin. In the kidney, the catabolism of glutamate increases the serum ammonium level while inhibiting the production of urea. HHS has been linked to childhood-onset epilepsy, learning disabilities, and seizures. To control this disorder, we found that EGCG and ECG inhibit GDH activity and block GDH-mediated insulin secretion. Expanding upon these results, the DeBerardinis group demonstrated that EGCG, via inhibition of GDH, can be used to target glioblastoma cells. Finally, the Blenis group demonstrated that EGCG can be used to kill tuberous sclerosis complex benign tumors via GDH inhibition.

The form of nitrogen nutrition affects many biochemical and physiological processes in plants, leading to markedly different growth responses. Many plant species have been characterized as ammonium tolerant or sensitive. The objective of this work was to assess the range of physiological adaptative responses involved in tolerance of ammonium nutrition at the intra-specific level. Pisum sativum L was selected as a model of tolerance for the study, and four varieties of agricultural importance (Snap-pea, Rondo, Tristar and Eclipse) were grown hydroponically with ammonium or nitrate (0.5, 2.5, 5.0 and 10.0 mM)as the sole nitrogen source. Physiological parameters including photosynthesis, respiration and ammonium contents were analyzed. Activities/expression of key enzymes of the nitrogen metabolism was also determined, for which novel antibodies against glutamate dehydrogenase and glutamine synthetase were obtained. Snap-pea was considered a reference plant for ammonium tolerance, since its biomass accumulation was not affected by nitrogen source. It avoided the physiological effects of ammonium through low growth potential, high respiratory rates and activity of the alternative pathway. A multivariate analysis of the results showed that each of the four varieties had distinct adaptative responses to ammonium nutrition, highlighting the flexibility of response to ammonium nutrition at the species level. A general activation of glutamate dehydrogenase (GDH) was observed, regardless of the degree of tolerance to ammonium, which may be due to GDH being part of a common stress response to carbon deficit rather than an ammonium tolerance mechanism. (C) 2010 Elsevier B.V. All rights reserved., This work was supported by the Spanish Ministry of Science and Innovation (grant nos. AGL2006-12792-CO2-01 and AGL2003-06571-CO2-01 [to PA-T] and AGL2007-64432/AGR [to JFM]), by the Government of Navarra (Res 57/2007 to JFM). MDD-V was supported by a doctoral Fellowship from the Basque Government, Spain.

Glucose homeostasis depends upon the appropriate release of insulin from pancreatic islet beta-cells. Postpandrial changes in circulating nutrient concentrations are coupled with graded release of stored insulin pools by the proportional changes in mitochondrial metabolism. The corresponding increased synthesis rates of both ATP and of anaplerotic metabolites have been shown to be mediators for nutrient-stimulated insulin secretion. Anaplerosis leads to the export of malate or citrate from the mitochondria, both of which can be recycled through metabolic pathways to reenter the Kreb's cycle. These metabolic cycles have the net effect of either transferring mitochondrial reducing equivalents to the cytosol, or of efficiently providing pyruvate to facilitate responsive changes in the Kreb's cycle flux in proportion to increased availability of glutamate and anaplerotic flux through glutamate dehydrogenase. Here, we describe siRNA knock-down and isotopic labeling strategies to evaluate the role of cytosolic and mitochondrial isoforms of malic enzyme in facilitating malate-pyruvate cycling in the context of fuel-stimulated insulin secretion.

Sirtuins (SIRT1-SIRT7) are a family of NAD(+)-dependent protein deacetylases that regulate cell survival, metabolism, and longevity. SIRT3 is localized to the mitochondria where it deacetylates several key metabolic enzymes: acetylcoenzyme A synthetase, glutamate dehydrogenase, and subunits of complex I and thereby regulates their enzymatic activity. SIRT3 is therefore emerging as a metabolic sensor that responds to change in the energy status of the cell via NAD(+) and that modulates the activity of key metabolic enzymes via protein deacetylation. Here we review experimental approaches that can be used in vitro and in vivo to study the role of acetylation in mitochondrial cell biology.

Publisher Summary This chapter focuses on a key enzyme of nitrogen metabolism in hyperthermophiles, glutamate dehydrogenase (GDH). GDHs are widely distributed enzymes involved in ammonia assimilation and catabolism of glutamate in microorganisms. GDH catalyses the reversible oxidative deamination of glutamate to 2-oxoglutarate and ammonia using NAD or NADP as cofactors. GDH represents an enzymatic link between major catabolic and biosynthetic pathways via the tricarboxylic acid (TCA) cycle intermediate 2-oxoglutarate. Analysis of GDH structures has provided the basis for comparative studies of extreme protein thermostability in this relatively complex enzyme. The chapter provides an overview of the purification and properties of thermostable GDHs and a review of the contributions that these studies have made to the understanding of the basis for exceptional enzyme stability.

Publisher Summary Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon belonging to the Euryarchaeota . Archaeoglobus fulgidus lacks a complete citric acid cycle, but the presence of enzymes required for the stepwise reductive formation of succinate from oxaloacetate and oxidative formation of 2-oxoglutarate from oxaloacetate and acetyl-CoA has been demonstrated, indicating the involvement of these enzymes in anabolism. Only two of these enzymes, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH), have so far been purified and characterized. Glutamate dehydrogenase (GDH), which generally interconnects the citric acid cycle with nitrogen assimilation through a reductive amination of 2-oxogluarate to glutamate, is absent in the A. fulgidus type strain (VC-16), but has been purified from an oil-field isolate (strain 7324), in which it is an abundant protein. This article describes the purification and the catalytic and molecular properties of IDH, GDH, and MDH from A. fulgidus . Their thermostability is also compared. The similarity of the primary structures of IDH and MDH with those of corresponding enzymes from other organisms is discussed.

Publisher Summary This chapter reviews the distribution of glutaminase in the brain and describes the glutamate synthesis and metabolism in glial cells. It discusses the role of glutamate and aspartate aminotransferase (AAT) in gamma-aminobutyric acid (GABAergic) neurons. Glutamate is synthesized directly from L-glutamine, 1-pyrroline-5-carboxylate (P5C) or ketoglutarate (2-oxoglutarate) in the central nervous system (CNS). The formation of glutamate from glutamine is an energy-saving process catalyzed by phosphate-activated glutaminase (PAG), which plays a major role in the production of transmitter glutamate. P5C is derived from ornithine through glutamic semialdehyde by the catalysis of ornithine-aminotransferase (OAT) or from proline by proline oxidase (PO), and then converted to glutamate by P5C dehydrogenase (P5CDH). α-Ketoglutarate is transformed to glutamate through reductive amination catalyzed by the reverse reaction of glutamate dehydrogenase (GDH). Ketoglutarate is also converted to glutamate through transamination reaction catalyzed by several aminotransferases such as aspartate aminotransferase (AAT) and alanine aminotransferase.