Glutamate Dehydrogenase Research Articles

Molecular pathways of osmoregulation in response to salinity stress in the gills of the scalloped spiny lobster (Panulirus homarus) within survival salinity

Scalloped spiny lobster (Panulirus homarus) aquaculture is the preferred strategy to resolve the conflict between supply and demand for lobster. Environmental conditions, such as salinity, are key to the success of lobster aquaculture. However, physiological responses of P. homarus to salinity stress have not been well studied. This study investigated the gill histology, osmoregulation and gill transcriptome of the early juvenile P. homarus (weight 19.04 ± 3.95 g) cultured at salinity 28 (control), 18, and 38 for 6 weeks. The results showed that the gill filaments of P. homarus exposed to low salinity showed severe separation of the cuticle and epithelial cells due to water absorption and swelling, as well as the dissolution and thinning of the cuticle and the rupture of the septum that separates the afferent and efferent channels. The serum osmolarity of P. homarus varied proportionately with external medium salinity and remained consistently above ambient osmolarity. The serum Na+, Cl−, K+, and Mg2+ concentrations P. homarus exhibited a pattern similar to that of serum osmolality, while the concentration of Ca2+ remained unaffected at salinity 18 but significantly increased at salinity 38. Gill Na+/K+-ATPase activity of P. homarus increased (p < 0.05) under the both salinity stress. Salinity 18 significantly increased Glutamate dehydrogenase (GDH) and Glutamicpyruvic transaminase (GPT) activity in the hepatopancreas of P. homarus (p < 0.05). According to transcriptome analysis, versus control group (salinity 28), 929 and 1095 differentially expressed genes (DEGs) were obtained in the gills of P. homarus at salinity 18 and 38, respectively, with these DEGs were mainly involved in energy metabolism, transmembrane transport and oxidative stress and substance metabolism. In addition, the expression patterns of 8 key DEGs mainly related to amino acid metabolism, transmembrane transport and oxidative stress were verified by quantitative real-time PCR (RT-qPCR). The present study suggests that salinity 18 has a greater impact on P. homarus than salinity 38, and P. homarus demonstrates effective osmoregulation and handle with salinity fluctuations (18 to 38) through physiological and functional adaptations. This study provides an improved understanding of the physiological response strategies of P. homarus facing salinity stress, which is crucial for optimizing aquaculture practices for this species.

Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

Serum Glutamate dehydrogenase activity enables sensitive and specific diagnosis of hepatocellular injury in humans.

Serum activities of alanine- and aspartate- aminotransferases (ALT and AST) are considered the "gold standard" biomarkers of hepatocyte injury in clinical practice and drug development. However, due to expression of ALT and AST in myocytes, the diagnosis of hepatocellular injury in patients with underlying muscle diseases, including drug-induced muscle injury, is severely limited. Thus, we proposed glutamate dehydrogenase (GLDH) as a liver-specific alternative to serum ALT and AST. In fact, our exploratory studies showed that GLDH has comparable performance to ALT for detecting hepatocyte injury without interference from concomitant muscle injury. Here, we report the results of studies confirming the reference intervals in a healthy human population and sensitivity and specificity of GLDH for detection of hepatocyte injury in human subjects. In human subjects, we could not perform liver biopsies due to ethical reasons, we also confirmed the relationship of GLDH and histopathologic lesions using 32 model toxicants in rats. Furthermore, we have shown that injury to tissues that are known to express appreciable levels of GLDH does not affect serum GLDH measurements, indicating excellent liver specificity of serum GLDH. Finally, we observed faster elimination of GLDH than ALT in humans, indicating that decreasing GLDH values could be considered an early sign of recovery. This study provides comprehensive evidence of excellent sensitivity and liver specificity of GLDH for diagnosis of hepatocellular injury, including evaluation of reference intervals that is essential for interpretation of serum GLDH in human subjects.

Effects of Replacing Fish Meal with Rubber Seed Cake on Growth, Digestive, Antioxidant and Protein Metabolism of Juvenile Asian Red-Tailed Catfish (Hemibagrus wyckioides).

An 8-week feeding trial was conducted to assess the impact of replacing fish meal (FM) with rubber seed cake (RSC) on growth performance, digestive enzyme activity of the gut, antioxidant capacity and protein metabolism-related parameters of plasma and liver in juvenile Asian red-tailed catfish (Hemibagrus wyckioides) with an average body weight of 3.21 g. Four isonitrogenous (41.08 ± 0.17) and isolipidic (10.07 ± 0.11) diets were formulated, incorporating RSC at a level of 0%, 15%, 30%, and 45% (w/w) to progressively replace FM. Dietary inclusion of 15-30% RSC did not significantly affect growth performance (p > 0.05). However, the replacement of FM with RSC resulted in linear reductions in intestinal trypsin and amylase activities, with a significant reduction in intestinal amylase activity observed at the 45% RSC inclusion level. Dietary inclusion of 30-45% RSC decreased plasma glutamate dehydrogenase and aspartate aminotransferase activities, but increased plasma adenosine monophosphate deaminase and hepatic γ-glutamyltransferase activities. This may indicate that the high substitution of FM by RSC may affect the homeostasis of amino acid metabolism. Dietary inclusion of 15% RSC did not affect serum and hepatic antioxidant enzyme activities; however, dietary inclusion of 45% RSC significantly increased plasma malondialdehyde level, but decreased catalase activity. Furthermore, dietary inclusion of 30-45% RSC significantly increased hepatic lactate dehydrogenase activity. These results demonstrated that replacing a portion of FM with RSC is feasible, and dietary inclusion of 15% RSC did not have significant adverse effects on growth performance, digestive function and antioxidant capacity of H. wyckioides.

Alterations in nitrogen metabolism caused by heavy metals in the acid-tolerant microalga Coccomyxa onubensis

The microalga Coccomyxa onubensis is an extremophile microorganism with a unique ecosystem (Río Tinto, Huelva, Spain) that contains high amounts of contaminants, including heavy metals, sulphates, and nitrates, in acidic environments (pH 2.5). The current work presents an evaluation of the capacity of Coccomyxa onubensis to assimilate different nitrogen sources under Cu2+, Cd2+, AsO33−, AsO43− and Hg2+ stress, and the metabolic implications of these stressors. The results showed that ammonium consumption was less affected than nitrate consumption when microalgae were cultivated with heavy metals (except cadmium). The activities of enzymes involved in nitrogen metabolism, such as nitrite reductase (NiR; EC:1.7.7.1), glutamine synthetase (GS; EC:6.3.2.1) and glutamate dehydrogenase (GDH; EC:1.4.1.2) were characterised to determine the Michaelis-Menten constant (Km) and optimal temperature and pH values, being 45, 40 and 60 °C and pH values of 7.5, 6.0 and 9.0 for NiR, GS, and GDH, respectively. The effects of different heavy metals on these enzymes were assessed at multiple levels, and the results showed that the enzymatic activity of NiR was downregulated, specially under copper stress, maintaining 23 % of control NiR activity at 2 mM Cu2+. The enzymatic activity of GS was upregulated at low concentrations under cadmium and mercury stress (115–120 % of control cultures GS activity at 25 μM Cd2+ and 50 nM Hg2+, respectively) and downregulated at high concentrations of these elements. GDH activity was upregulated in the presence of Cu2+, Cd2+, and Hg2+, with increases up to 192, 155 and 154 % at 1 mM Cu2+, 300 μM Cd2+, and 250 nM Hg2+, respectively. These results provide a better explanation of the effects of heavy metal stress on N metabolism in Coccomyxa onubensis, which may be used as a model eukaryotic organism of the Tinto River acidophilic ecosystem.

Metabolic Checkpoints in Rheumatoid Arthritis

BackgroundRheumatoid Arthritis is a systemic autoimmune disease affecting 0.5-1% of the population. Despite a growing therapeutic armamentarium, RA remains incurable, and many patients suffer significant morbidity over time. The strongest genetic risk derives from HLA class II polymorphisms, implicating T cells as pathogenic drivers. Innate immune cells, e.g. monocytes and macrophages (Mⱷ) contribute to chronic tissue inflammation through an array of pro-inflammatory functions but also present antigen to autoreactive T cells. Differentiation, survival, and effector functions of both T cells and Mⱷ are ultimately controlled by their bioenergetic and biosynthetic programs, identifying cellular metabolism as a critical disease mechanism in RA. ObjectivesSummarize current knowledge about metabolic conditions in the RA joint and disease-relevant metabolic circuits shaping the effector repertoire of RA T cells and Mⱷ. ResultsThe rheumatoid joint is a glucose deplete tissue environment, selecting for invading immune cells that can survive on non-glucose fuel sources. Inflamed synovium instead offers the amino acid glutamine and RA CD4+ T cells and RA Mⱷ rely on glutamine and glutamate to support their pathogenic functions. The metabolic hallmark of RA T cells is their low mitochondrial performance, resulting in low ATP production, low generation of reactive oxygen species (ROS) and low availability of tricarboxylic acid (TCA) cycle intermediates, all shifting RA T cells towards autoreactivity. The underlying defect stems from insufficient repair of mitochondrial DNA (mtDNA). Functional consequences include reversal of the TCA cycle, accumulation of citrate and lack of malate production. Excessive citrate promotes cytoskeletal hyperacetylation, creating hypermigratory and tissue-invasive T cells. Surplus acetyl-CoA supports lipid droplet formation and lipotoxicity. Lack of malate production disrupts the malate-aspartate shuttle, restricts recovery of cytosolic NAD and drives the endoplasmic reticulum (ER) into expansion. The bioenergetically stressed ER accumulates TNF mRNA and turns RA T cells into TNF superproducers. ATP low production renders RA T cells susceptible to cell death, depositing highly inflammatory mtDNA in the tissue. Mitochondrial deficiency leads to a slowdown in glycolysis and pyruvate processing, such that RA CD4+ T cells shunt glucose towards the pentose phosphate pathway to support nucleotide synthesis and clonal proliferation. Metabolically deprived CD4+ T cells partner with Mⱷ that have highly functional mitochondria. A hallmark of RA Mⱷ is the high expression of the DNA binding protein RFX5, which co-ordinates adaptations to metabolic needs with function. RFX5 upregulates HLA-DR expression and induces the glutaminolytic enzyme glutamate dehydrogenase 1 (GLUD1), providing bioenergetic resources for antigen presentation and survival in the tissue. In essence, RA CD4+ T cells and Mⱷ function in a metabolically challenging environment and rewire their cellular metabolism to survive. Metabolic adaptations promote immunostimulation and tissue inflammation, triggering and sustaining rheumatoid synovitis.

Effects of Water–Nitrogen Interaction on Sandy Soil, Physiology, and Morphology of Tall Fescue (Festuca arundinacea Schreb) Turf

The soil water and nitrogen (N) levels are the important factors affecting turfgrass growth. However, the impact of the water–N interaction on tall fescue (Festuca arundinacea Schreb) in terms of the N metabolism and plant morphology remains uncertain. Therefore, the objective of this study was to investigate the impacts of different N and water levels on the physiological and morphological responses of tall fescue. The experiment was designed with N (N0, N2, and N4 representing N application rates of 0, 2, and 4 g m–2, respectively) and irrigation [W1, W2, W3, W4, and W5 representing field water capacities (FWCs) of 90~100%, 75~85%, 60~70%, 45~55%, and 30~40%, respectively] treatments, and the relevant indexes of the soil water content and soil NH4+–N and NO3−–N levels as well as the physiology and morphology of the tall fescue were determined. The results demonstrated significant changes in the contents of soil water (SWC) and N and the physiological and morphological indexes, except for the enzymes related to N metabolism, including nitrite reductase (NiR), glutamate dehydrogenase (GDH), and glutamate synthetase (GOGAT). The water stress significantly enhanced the water and N use efficiencies (WUE and NUE), except the NUE in the W5 treatment. The N stress significantly influenced the SWC, soil NO3−–N content, and physiological and morphological indexes, excluding malondialdehyde, NiR, GOGAT, and above- (AGB) and below-ground biomass, resulting in the increased WUE and NUE. The application of a low N rate effectively alleviated the detrimental impacts of water stress on the SWC and glutamine synthetase activity. In conclusion, W2 and N2 are deemed more appropriate treatments for the low-maintenance measures of tall fescue turf. Among all the treatments, N2W2 is recommended as the optimal water–N interaction treatment due to its ability to conserve resources while still ensuring high turf quality.

When and What to Test for Diarrhea: Focus on Stool Testing.

To review stool diagnostic tests in acute and chronic diarrhea. Narrative review of published literature. (A). In acute diarrhea, stool tests are indicated when there is strong pre-test probability of infectious etiology or C. difficile infection (CDI) suggested by >3 unformed bowel movements per 24h, symptoms lasting >7 days, and circumstances that are suggestive of infection. Several commercially available rapid tests for bacterial, viral or protozoal infections may be offered in addition to traditional methods (e.g. culture, microscopy), and provide a result within 6 hours. For CDI infection, a highly sensitive test such as glutamate dehydrogenase test is required; however, this does not distinguish infection from carrier state. That differentiation requires specialized nucleic acid amplification test (for toxin B) or enzyme immunoassays for toxin A or B, which are unfortunately not generally offered by microbiology labs. (B). Chronic diarrhea may result from inflammatory, fatty, osmotic or secretory causes; the commonest causes are IBS-D/functional diarrhea. Current recommendations in societal guidelines or clinical practice updates regarding stool tests in IBS-D/functional diarrhea in the absence of alarm symptoms include testing for giardia, calprotectin, fecal immunochemical test (FIT), and bile acid diarrhea (BAD). Comprehensive stool biochemical analyses (osmolality, pH, electrolytes) differentiate osmotic from secretory diarrhea and identify laxative abuse. Specific stool diagnostic tests for BAD and exocrine pancreatic insufficiency can lead to specific diagnosis and treatments. Surrogate markers associated with high fecal output and rapid transit in chronic diarrhea are stool form and colonic transit. Fecal testing is still very relevant in the practice of gastroenterology and deserves introduction of advanced microbiological and biochemical tests.

GDH1-catalytic glutaminolysis feedback activate EGFR/PI3K/AKT pathway and reprogram glioblastoma metabolism.

Glutamine is an important nutriment for cancer cell growth that provides biological sources for nucleic acid and fatty acid synthesis, but the role of glutaminolysis in signal transduction and glioblastoma (GBM) progression remains little known. Knockdown and overexpression cells were obtained to explore the functional roles of GDH1 in cell proliferation, tumor formation and aerobic glycolysis. RNA-seq, Chromatin immunoprecipitation, luciferase assay and western blot were performed to verify the regulation of EGFR-AKT pathway by the glutamate dehydrogenase 1 (GDH1, also known as GLUD1) and KDM6A. Metabolite-level measurements and Seahorse Assay were performed to assess the functional role of GHD1 in reprogramming glycolysis. Here, we report that GDH1 catalytic glutaminolysis is essential for GBM cell line proliferation and brain tumorigenesis even in high-glucose conditions. Glutamine is metabolized through glutaminolysis to produce α-ketoglutarate (α-KG). We demonstrate that glutamine in combination with leucine activates mammalian TORC1 by enhancing glutaminolysis and α-KG production. α-KG increases the transcription of PDPK1 by reducing the suppressive histone modification H3K27me3, and then promotes the activation of PI3K/AKT/mTOR pathway. This transcriptional activation induced by α-KG requires histone demethylase KDM6A, which is a 2-oxoglutarate oxygenase that plays important roles in converting α-KG to succinate. Furthermore, we show that GDH1-catalytic glutaminolysis also increases the expression of HK2 and promotes glycolysis in high-glucose condition dependent on KDM6A-mediated demethylation of H3K27. These findings suggest a novel function of glutaminolysis in regulation of signal transduction and metabolism reprograming, provide further evidence for unique role of glutaminolysis in GBM progression.

Computer-Aided Flexible Loops Engineering of Glutamate Dehydrogenase for Asymmetric Synthesis of Chiral Pesticides l-phosphinothricin.

The access to the enantiopure noncanonical amino acid l-phosphinothricin (l-PPT) by applying biocatalysts is highly appealing in organic chemistry. In this study, a NADH-dependent glutamate dehydrogenase from Lachnospiraceae bacterium (LbGluDH) was chosen for the asymmetric synthesis of l-PPT. Three flexible loops undergoing big conformational shifts during the catalysis were identified and rationally engineered following the initial mutagenesis. The enzyme's specific activity toward the key precursor of l-PPT, 2-oxo-4-[(hydroxy) (methyl) phosphinyl] butyric acid (PPO), was improved from negligible to 9 U/mg, and the Km value was reduced to 17 mM. The computational analysis showed that the modified loops broadened the enzyme's narrow tunnels, allowing the substrate to access the binding pocket and get closer to the crucial residue D165, thereby enhancing the catalytic process. Utilizing the variant as the catalyst, the preparation of l-PPT achieved a 100% conversion rate within 60 min, coupled with a stereoselectivity exceeding 99.9%, demonstrating its practical capacity for industrial application. Similar enhancement in catalytic activity was obtained applying the same strategy to a typical NADH-dependent GluDH from Pyrobaculum islandicum (PisGluDH), indicating the effectiveness of our strategy for the protein engineering of GluDHs targeted to the biosynthesis of unnatural compounds.

Pyruvate stimulates transamination of leucine into α-ketoisocaproic acid and supports 3-methylbutanal production by Lactococcus lactis.

To investigate the effect of pyruvate and glucose on leucine transamination and 3-methylbutanal production by Lactococcus lactis, including the comparison with cells possessing glutamate dehydrogenase (GDH) activity. L. lactis cells were incubated in chemically defined medium (CDM) with the pH controlled at 5.2 to mimic cheese conditions. Pyruvate supplementation stimulated the production of the key flavour compound 3-methylbutanal by 3 to 4 times after 72 hours of incubation. Concurrently, alanine production increased, demonstrating the involvement of pyruvate in transamination reactions. Glucose-metabolising cells excreted α-ketoisocaproic acid and produced even 3 times more 3-methylbutanal after 24 hours than pyruvate-supplemented cells. Conjugal transfer technique was used to transfer the plasmid pGdh442 carrying the gdh gene encoding for GDH to L. lactis. Introducing GDH did not stimulate the excretion of α-ketoisocaproic acid and the production of 3-methylbutanal. These results demonstrate that Lactococcus uses pyruvate to transaminate leucine into α-ketoisocaproic acid which supports 3-methylbutanal production. Surprisingly, GDH activity did not stimulate leucine transamination and 3-methylbutanal production.

Elevated peripheral glutamate and upregulated expression of NMDA receptor NR1 subunit in insomnia disorder.

The present study explored the serum glutamate (Glu), glutamine (Gln), glutamic acid dehydrogenase (GAD) concentrations and the mRNA expression levels of the N-methyl-D-aspartate receptor (NMDAR) NR1 subunit in the peripheral blood of patients with insomnia disorder (ID). To our knowledge, this is the first study showing an increase in the mRNA expression levels of the NMDAR NR1 subunit in patients with ID. This study included 30 ID patients and 30 matched healthy controls. We investigated the demographic and illness information and assessed subjective sleep quality using the Pittsburgh Sleep Quality Index. The Hamilton Depression Scale-17 and Hamilton Anxiety Scale were used to evaluate the patients' symptoms of depression and anxiety, respectively. The quantifications of Glu, Gln and GAD concentrations were performed by Enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to detect the mRNA expression levels of the NMDAR NR1 subunit in peripheral blood. Compared with the healthy control group, the serum Glu concentrations and the mRNA expression levels of the NMDAR NR1 subunit in the ID group were significantly higher. However, there was no significant difference in Gln and GAD between the two groups. The receiver operating characteristic (ROC) analysis showed that the mRNA expression levels of the NMDAR NR1 subunit could distinguish ID patients from healthy individuals (area under the curve: 0.758; sensitivity: 73.3%; specificity: 76.7%). A negative correlation was found between the mRNA expression levels of the NMDAR NR1 subunit for age, total duration of illness, and age of first onset in the ID group, whereas a positive correlation was detected for daytime dysfunction. Glutamatergic neurotransmission was abnormal in ID patients. Additionally, the mRNA expression levels of the NMDAR NR1 subunit appeared to have potential as a clinical biomarker for ID. However, the sample size of our study was limited, and future studies with larger sample sizes are needed to further validate and explore the mechanisms involved and to assess the reliability of the biomarker.

7354 A Case of Continuous Glucose Monitoring in Children with Glutamate Dehydrogenase Hyperinsulinism

Abstract Disclosure: K. Kim: None. E. Mun: None. C. Jung Eun: None. R. Lee: None. K. Hae Soon: None. A Case of Continuous Glucose Monitoring in Children with Glutamate Dehydrogenase(GLUD1) Hyperinsulinism Background: Continuous glucose monitoring (CGM) is increasingly being used in pediatric patients with diabetes. CGM can help prevent hypoglycemia and support the management of hypoglycemic problems. Congenital hyperinsulinism is a rare condition in which hypoglycemia occurs due to hyperinsulinemia caused by the dysregulation of insulin secretion, and it can lead to complications such as developmental delay and seizures. Case presentation: A 14 months years old, male infant was referred to the pediatric emergency room representing symptoms of GTC type seizure during 2-3 minutes and hypoglycemia. He had no perinatal problems with no significant family history. During seizure, He was found to have a blood glucose of 39mg/dL, ammonia of 183μmol/L, insulin of 28.3 mcg/mL and ketone 203.4μmol/L. C-peptide was 1.4 ng/mL (≥ 0.5ng/mL) and free fatty acid was 0.492mmol/L (&amp;lt;1.7mmol/L) for evaluation. He was thought to have GDH hyperinsulinism. Blood glucose level was followed up with feeding frequency and protein restricted diet. Congenital hyperinsulinism genetic test was requested. Brain CT showed normal. EEG revealed no focal abnormalities, no persistent asymmetries. CGM was used in patient. Diazoxide was started with 5mg/kg/day in 2 divided doses and then blood glucose level was observed below 60mg/dL 3-4 times for day. Complete blood counts and uric acid levels were measured at baseline and 5 days after starting diazoxide therapy, and thereafter increase to 6.25mg/kg/day Conclusions: In children with GDH hyperinsulinism, the utilization of CGM can serve as a tool to assess the impact of treatment before and after medication therapy, as well as also aiding in hypoglycemia prevention. Presentation: 6/2/2024

A-084 Performance evaluation of Sentinel Diagnostics’ Ammonia Ultra assay on Beckman Coulter AU/DxC AU Systems

Abstract Background The analytical performances of the Ammonia Ultra assay for in vitro quantitative enzymatic determination of Ammonia in human plasma have been evaluated on Beckman AU/DxC AU Systems. Ammonia, derived from the amino acids catabolism and from the action of intestinal bacteria on dietary proteins, is converted to non-toxic urea by the liver. An excess of ammonia can have toxic effects on the Central Nervous System with possible neurological disturbances. Elevated ammonia levels can also be observed in severe liver failure as may occur in Reye’s syndrome, viral hepatitis or cirrhosis. Methods Ammonia, in the presence of glutamate dehydrogenase (GLDH), combines with alpha-ketoglutarate and NADH to yield glutamate and NAD+. The decrease in 340 nm absorbance (NADH → NAD+) is proportional to the ammonia level in the examined plasma. The reagent contains excess of lactate dehydrogenase (LDH) to rapidly reduce endogenous pyruvate and avoid its interference with the assay system. Performances evaluation have been done following current CLSI guidelines protocols. Results 3 calibrator lots and 2 reagent lots have been used for testing. For each test and among different combinations of reagents and calibrators, worst case has been reported in the table. Conclusions Analytical performances of Ammonia Ultra assay on Beckman AU/DxC AU Systems meet the requirements for its use as quantitative determination of Ammonia in human plasma and make this assay very suitable for the routine measurement of this analyte.

Cucurbita Pepo L. Seed Oil Modulates Dyslipidemia and Neuronal Dysfunction in Tramadol-Induced Toxicity in Wistar Albino Rats.

Objective: The modulating effects of Cucurbita pepo seed oil (CPSO) on dyslipidemia and neuronal dysfunction in tramadol toxicity were studied. Methods: Fifty-six albino rats were divided into seven groups of eight rats each after a 2-week acclimatization period. All animals had unrestricted access to water and feed, and treatments were administered orally once daily for 42 days. Glutamate dehydrogenase and glutaminase activities were assessed using brain homogenate, while lipid profiles were analyzed in serum samples. Results: Tramadol toxicity was evidenced by significant (P < 0.05) increases in brain glutamate dehydrogenase along with significant (P < 0.05) decreases in the activities of glutaminase in the group administered only tramadol. Also, serum levels of total cholesterol, LDL-C and triglycerides also increased significantly (P < 0.05) following administration of tramadol with decreased level of HDL-C (P < 0.05). However, treatment with CPSO significantly restored the activities and levels of the altered biochemical parameters in a dose-dependent manner. The results of the biochemical investigation using the lipid profile and the enzymes of glutamate metabolism were corroborated by the results obtained from the histopathological examination of the brain. Conclusion: The results of this study therefore suggest that tramadol-induced dyslipidemia and neuronal dysfunction be managed and prevented by the administration of Cucurbita pepo seed oil.

Lateral flow immunoassay for simultaneous detection of C. difficile, MRSA, and K. pneumoniae

Mainly performed within a rapid diagnostic tests company, a lateral flow (LF) system using gold nanoparticles (AuNPs) as transducers is presented able to detect three bacteria of interest, of relevance for antimicrobial resistance (AMR): Clostridioides difficile, methicillin-resistant Staphylococcus aureus (MRSA), and Klebsiella pneumoniae, with a limit of detection of 25 ng/mL of glutamate dehydrogenase (GDH) for C. difficile, 36 ng/mL of penicillin-binding protein 2a (PBP2a) for MRSA, and 4 × 106 CFU/mL for K. pneumoniae. The system showed good results with bacteria culture samples, is user-friendly, and suitable for rapid testing, as the results are obtained within 15 min.Graphical

Arabidopsis GDH1 and GDH2 genes double knock-out results in a stay-green phenotype during dark-induced senescence.

Yellowing is the first visually observable sign of plant leaf senescence. We found that Arabidopsis double knockout mutant gdh1gdh2 for genes of NAD(H)-dependent glutamate dehydrogenase retains green color of the leaves (stay-green phenotype) during a dark-induced senescence, in contrast to wild-type plants, whose leaves turn yellow. When the gdh1gdh2 plants are exposed to the dark more than four days, they demonstrate slower chlorophyll degradation than in the wild-type plants under the same conditions, as well as dysregulation of chlorophyll breakdown genes encoding chlorophyll b reductase, Mg-dechelatase, pheophytinase and pheophorbide a oxygenase. The slowed degradation of chlorophyll b in gdh1gdh2 plants significantly alters the chlorophyll a/b ratio. Ion leakage in the mutant plants increases significantly from four to eight days in the darkness, correlating with their premature death during this period. The discovered facts suggest a functional connection between activity of NAD(H)-dependent glutamate dehydrogenase and dark-induced senescence progress in Arabidopsis.

Evaluating the Effects of Ketamine on Serum Glutamate Dehydrogenase, Glutamine Synthetase and Asparagine Synthetase Levels in Patients with Major Depressive Disorder Before and After Ketamine Treatment

Introduction: It has been revealed that major depressive disorder (MDD)is a common and debilitating psychiatric disorder. Dysfunctional enzymes involved in neurotransmission glutamate dehydrogenase (GDH), Glutamine synthetase (GS) and Asparagine synthetase (ASNS) may underlie the pathophysiology of MDD. The present project aimed to explore the effects of Ketamine on GDH, GS and ASNS in MDD patients. This study provides evidence of interactions between ketamine and GDH, ASNS and GS levels in patients with MDD. Materials and Methods: Patients with MDD are referred to the psychiatric ward of Shaheed Yahyanejad Hospital for regular follow-up. Patients diagnosed with MDD were based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), structured clinical interviews, as well as the severity of depression based on clinical criteria determined by a specialist physician. Serum samples from MDD patients before and after ketamine administration were taken to examine the changes in GDH, ASNS and GS levels. In this project, 29 patients with MDD therapy were evaluated with ketamine (0.75 mg/kg). Results: Our study showed that after administrating ketamine, the level of ASNS and Glutamate dehydrogenase GDH in patients with MDD was reduced compared to pre-treatment. However, the level of GS was increased compared to before treatment. The results show that ketamine occurs at the metabolism level in MDD patients. Also, our results demonstrated that GDH, ASNS, and GS levels can be measured to evaluate the effect of Ketamine on MDD patients. Conclusions: Patient success has improved depression after two months of administrating ketamine. Marginally, more than 80% of patients diagnosed with MDD treated with ketamine showed complete remission. Current results may be helpful in understanding the mechanisms responsible for ketamine’s clinical efficacy.

Biochemical and Epigenetic Regulation of Glutamate Metabolism in Maize (Zea mays L.) Leaves under Salt Stress.

The effect of salt stress (150 mM NaCl) on the expression of genes, methylation of their promoters, and enzymatic activity of glutamate dehydrogenase (GDH), glutamate decarboxylase (GAD), and the 2-oxoglutarate (2-OG)-dehydrogenase (2-OGDH) complex was studied in maize (Zea mays L.). GDH activity increased continuously under salt stress, being 3-fold higher after 24 h. This was accompanied by the appearance of a second isoform with lower electrophoretic mobility. The expression of the Gdh1 gene strongly increased after 6-12 h of incubation, which corresponded to the demethylation of its promoter, while Gdh2 gene expression slightly increased after 2-6 h and then decreased. GAD activity gradually increased in the first 12 h, and then returned to the control level. This corresponded to the increase of Gad expression and its demethylation. Salt stress led to a 2-fold increase in the activity of 2-OGDH during the first 6 h of NaCl treatment, then the activity returned to the control level. Expression of the genes Ogdh1 and Ogdh3 peaked after 1-2 h of incubation. After 6-8 h with NaCl, the expression of these genes declined below the control levels, which correlated with the higher methylation of their promoters. We conclude that salt stress causes a redirection of the 2-OG flux to the γ-aminobutyric acid shunt via its amination to glutamate, by altering the expression of the Gdh1 and Gdh2 genes, which likely promotes the assembly of the native GDH molecule having a different subunit composition and greater affinity for 2-OG.

Integration of metabolomic and transcriptomic analyses reveals novel regulatory functions of the ChREBP transcription factor in energy metabolism.

Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.