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1
Tomita, Takeo, Takashi Miyazaki, Junichi Miyazaki, Tomohisa Kuzuyama, and Makoto Nishiyama. "Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus." Microbiology 156, no. 12 (December 1, 2010): 3801–13. http://dx.doi.org/10.1099/mic.0.042721-0.
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An extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes, gdhA and gdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA–GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA–GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1 mM leucine elevated the GDH activity of the co-purified GdhA–GdhB by 974 and 245 % for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a hetero-oligomeric GDH that can be regulated allosterically.
2
Coschigano, P. W., S. M. Miller, and B. Magasanik. "Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 9 (September 1991): 4455–65. http://dx.doi.org/10.1128/mcb.11.9.4455.
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We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.
3
Coschigano, P. W., S. M. Miller, and B. Magasanik. "Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 9 (September 1991): 4455–65. http://dx.doi.org/10.1128/mcb.11.9.4455-4465.1991.
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We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine. This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified. This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources. Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized. Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.
4
Janes, Brian K., Pablo J. Pomposiello, Ana Perez-Matos, David J. Najarian, Thomas J. Goss, and Robert A. Bender. "Growth Inhibition Caused by Overexpression of the Structural Gene for Glutamate Dehydrogenase (gdhA) fromKlebsiella aerogenes." Journal of Bacteriology 183, no. 8 (April 15, 2001): 2709–14. http://dx.doi.org/10.1128/jb.183.8.2709-2714.2001.
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ABSTRACT Two linked mutations affecting glutamate dehydrogenase (GDH) formation (gdh-1 and rev-2) had been isolated at a locus near the trp cluster in Klebsiella aerogenes. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The gdhA gene from K. aerogenes was cloned and sequenced, and an insertion mutation was generated and shown to be linked to trp. A region ofgdhA from a strain bearing gdh-1 was sequenced and shown to have a single-base-pair change, confirming that the locus defined by gdh-1 is the structural gene for GDH. Mutants with the same phenotype as rev-2 were isolated, and their sequences showed that the mutations were located in the promoter region of the gdhA gene. The linkage of gdhA totrp in K. aerogenes was explained by postulating an inversion of the genetic map relative to other enteric bacteria. Strains that bore high-copy-number clones of gdhAdisplayed an auxotrophy that was interpreted as a limitation for α-ketoglutarate and consequently for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy-number clones of the enzymatically inactive gdhA1 allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the α-ketoglutarate supply or reduce the succinyl-CoA requirement relieved the auxotrophy.
5
Daningsih, Entin, D. L. Coffey, J. Logan, and C. A. Mullins. "GROWTH STUDIES WITH SNAP BEANS TO PREDICT HARVEST." HortScience 25, no. 9 (September 1990): 1140e—1140. http://dx.doi.org/10.21273/hortsci.25.9.1140e.
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Studies were initiated in 1989 to characterize phonological events with corresponding growth and development phenomena of `Eagle' and `Provider' snap beans (Phaseolus vulgaris L.) Ten plantings at approximately 15 day intervals were made at Knoxville, TN from April 17 through July 27. Days to reach growth stages V0 thru R7 were recorded for each cultivar for each planting date. Air temperature, total radiant energy, wind speed and relative humidity were recorded hourly throughout the 171 day test period. Growing degree days (GDD) computed by 8 methods and growing degree hours (GDH) computed by 2 methods were regressed against plant developmental stages. GDD and GDH, along with pod size and pod fiber content, will be discussed as possible indices for predicting harvest maturity. With the methods used to calculate heat summation in this study, GDD and GDH from planting to pod maturity ranged from approximately 550 to 975 and 9,700 to 20,000, respectively.
6
Lu, Chung-Dar, and Ahmed T. Abdelal. "The gdhB Gene of Pseudomonas aeruginosaEncodes an Arginine-Inducible NAD+-Dependent Glutamate Dehydrogenase Which Is Subject to Allosteric Regulation." Journal of Bacteriology 183, no. 2 (January 15, 2001): 490–99. http://dx.doi.org/10.1128/jb.183.2.490-499.2001.
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ABSTRACT The NAD+-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encodinggdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, andCaulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) fromSaccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.
7
Schroeder, Jill M., Wenlin Liu, and Norman P. Curthoys. "pH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+cells." American Journal of Physiology-Renal Physiology 285, no. 2 (August 2003): F258—F265. http://dx.doi.org/10.1152/ajprenal.00422.2002.
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During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3′-untranslated region (3′-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds ζ-crystallin/NADPH:quinone reductase (ζ-crystallin) with high affinity and functions as a pH-response element. RNA EMSAs established that ζ-crystallin also binds to the full-length 3′-UTR of GDH mRNA. This region contains four eight-base sequences that are 88% identical to one of the two GA AREs. Direct binding assays and competition studies indicate that the two individual eight-base AREs from GA mRNA and the four individual GDH sequences bind ζ-crystallin with different affinities. Insertion of the 3′-UTR of GDH cDNA into a β-globin expression vector (pβG) produced a chimeric mRNA that was stabilized when LLC-PK1-F+cells were transferred to acidic medium. A pH-responsive stabilization was also observed using a βG construct that contained only the single GDH4 ARE and a destabilizing element from phospho enolpyruvate carboxykinase mRNA. Therefore, during acidosis, the pH-responsive stabilization of GDH mRNA may be accomplished by the same mechanism that affects an increase in GA mRNA.
8
Orlandi, Fabio, Bruno Romano, and Marco Fornaciari. "Relationship between Flowering and Heat Units to Analyze Crop Efficiency of Olive Cultivars Located in Southern Italy." HortScience 40, no. 1 (February 2005): 64–68. http://dx.doi.org/10.21273/hortsci.40.1.64.
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The relationship between heat units trends and reproductive development in olive (Olea europaea, L.) was studied over a 3-year period (1999-2001) in 15 areas in the southern Italian regions of Campania, Calabria, Puglia, and Sicily. Heat units were calculated using GDH and GDD formulas and the flowering phases in the olive groves were studied using volumetric pollen traps that aspirate fixed quantities of air. With this method, the olive pollen release and flowering trends were determined. The main objective of the study was to correlate the spring heat unit amounts and the phases of maximum pollen emission with the date of flowering. Moreover, the ranges of GDH and GDD in the different study areas were calculated to identify maps of olive pollen release.
9
Hohnholt, Michaela C., Vibe H. Andersen, Jens V. Andersen, Sofie K. Christensen, Melis Karaca, Pierre Maechler, and Helle S. Waagepetersen. "Glutamate dehydrogenase is essential to sustain neuronal oxidative energy metabolism during stimulation." Journal of Cerebral Blood Flow & Metabolism 38, no. 10 (June 16, 2017): 1754–68. http://dx.doi.org/10.1177/0271678x17714680.
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The enzyme glutamate dehydrogenase (GDH; Glud1) catalyzes the (reversible) oxidative deamination of glutamate to α-ketoglutarate accompanied by a reduction of NAD+ to NADH. GDH connects amino acid, carbohydrate, neurotransmitter and oxidative energy metabolism. Glutamine is a neurotransmitter precursor used by neurons to sustain the pool of glutamate, but glutamine is also vividly oxidized for support of energy metabolism. This study investigates the role of GDH in neuronal metabolism by employing the Cns- Glud1−/− mouse, lacking GDH in the brain (GDH KO) and metabolic mapping using 13C-labelled glutamine and glucose. We observed a severely reduced oxidative glutamine metabolism during glucose deprivation in synaptosomes and cultured neurons not expressing GDH. In contrast, in the presence of glucose, glutamine metabolism was not affected by the lack of GDH expression. Respiration fuelled by glutamate was significantly lower in brain mitochondria from GDH KO mice and synaptosomes were not able to increase their respiration upon an elevated energy demand. The role of GDH for metabolism of glutamine and the respiratory capacity underscore the importance of GDH for neurons particularly during an elevated energy demand, and it may reflect the large allosteric activation of GDH by ADP.
10
Hynes, M. J. "The Effects of the Carbon Source on Glutamate Dehydrogenase Activities in Aspergillus nidulans." Microbiology 81, no. 1 (January 1, 2000): 165–70. http://dx.doi.org/10.1099/00221287-81-1-165.
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The NADP-specific glutamate dehydrogenase (NADP-GDH) activity of Aspergillus nidulans was rapidly lost from cultures starved for a carbon source. This loss of NADP-GDH was blocked by protein synthesis inhibitors. Glutamate repressed NADP-GDH but did not cause rapid loss of activity. Since NADP-GDH is involved in the participation of ammonium in the regulation of nitrogen metabolism, the loss of NADP-GDH activity accompanying carbon starvation may be important in the interaction between carbon and nitrogen metabolism. Increased NAD specific glutamate dehydrogenase activity (NAD-GDH) was observed when mycelium was transferred to medium lacking glucose. The increase in NAD-GDH activity was greatest when glutamate was present. Protein synthesis inhibitors did not prevent this increase in activity. Two mutants, amdT102 and amdT19, which are altered in regulation of nitrogen metabolism, are similar to the wild-type strain with regard to regulation of NADP-GDH and NAD-GDH.
11
Kennedy, JR, Z. Wyszomirska-Dreher, Y. T T chan, and JW Chen. "Properties of Antisera to Glutamate Dehydrogenase from Nitrogen-fixing Lupin Nodules." Australian Journal of Biological Sciences 38, no. 1 (1985): 51. http://dx.doi.org/10.1071/bi9850051.
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Antibody prepared in rabbit to lupin (Lupinus luteus) nodule glutamate dehydrogenase (GDH) crossreacted with all six isozymes of GDH isolated from lupin nodules. Rocket immunoelectrophoresis showed that the antisera were also strongly cross-reactive with GDH from other parts of the lupin plant and from the roots and stems of other leguminous plants and wheat, but not with GDH of Rhizobium lupini, lupin bacteroids or bovine liver. This confirms the exclusively plant origin of lupin nodule cytosolic GDH. Enzyme activity, determined spectrophotometrically, was strongly inhibited by the antibody. Substrates and modifiers of GDH did not influence the degree of this inhibition, indicating that the antiserum should be an effective reagent for study of the localization of GDH in plants.
12
Timmerman, M., R. B. Wilkening, and T. R. H. Regnault. "Induction of Glutamate Dehydrogenase in the Ovine Fetal Liver by Dexamethasone Infusion during Late Gestation1." Experimental Biology and Medicine 228, no. 1 (January 2003): 100–105. http://dx.doi.org/10.1177/153537020322800114.
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Glucocorticoids near term are known to upregulate many important enzyme systems prior to birth. Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes both the reversible conversion of ammonium nitrogen into organic nitrogen (glutamate production) and the oxidative deamination of glutamate resulting in 2-oxoglutarate. The activity of this enzyme is considered to be of major importance in the development of catabolic conditions leading to gluconeogenesis prior to birth. Ovine hepatic GDH mRNA expression and activity were determined in near-term (130 days of gestation, term 147 ± 4 days) control and acutely dexamethasone-treated (0.07 mg–1 hr–1 for 26 hr) fetuses. Dexamethasone infusion had no effect on placental or fetal liver weights. Dexamethasone infusion for 26 hr significantly increased hepatic GDH mRNA expression. This increased GDH mRNA expression was accompanied by an increase in hepatic mitochondrial GDH activity, from 30.0 ± 7.4 to 58.2 ± 8.1 U GDH/U CS (citrate synthase), and there was a significant correlation between GDH mRNA expression and GDH activity. The generated ovine GDH sequence displayed significant similarity with published human, rat, and murine GDH sequence. These data are consistent with the in vivo studies that have shown a redirection of glutamine carbon away from net hepatic glutamate release and into the citric acid cycle through the forward reaction catalyzed by GDH, i.e., glutamate to oxoglutarate.
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Srivastava, Gautam, Mohan Pal, Suneet Kaur, and Ravinder S. Jolly. "A highly efficient designer cell for enantioselective reduction of ketones." Catalysis Science & Technology 5, no. 1 (2015): 105–8. http://dx.doi.org/10.1039/c4cy01017e.
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A highly efficient designer cell, surf-crs-gdh, which coexpresses carbonyl reductase (crs) and glucose dehydrogenase (gdh) on the cell surface, has been constructed and its enzyme activities were compared with those of the corresponding cell, cyto-crs-gdh, which coexpresses crs and gdh in cytoplasm.
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Mounzer, Oussama H., Wenceslao Conejero, Emilio Nicolás, Isabel Abrisqueta, Yelitza V. García-Orellana, Luis M. Tapia, Juan Vera, Jose M. Abrisqueta, and Maria del Carmen Ruiz-Sánchez. "Growth Pattern and Phenological Stages of Early-maturing Peach Trees Under a Mediterranean Climate." HortScience 43, no. 6 (October 2008): 1813–18. http://dx.doi.org/10.21273/hortsci.43.6.1813.
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The phenological stages of early-maturing peach trees were described using the traditional nomenclature of Baggiolini and according to the BBCH General Scale. The heat requirement of each stage was calculated as growing degree hours (GDH) and growing degree days (GDD). The annual growth pattern of trunk, shoot, and fruit was also studied. After dormancy breaking involving 225 chilling units, this early peach cultivar required ≈6244 GDH to reach full bloom and 27106 GDH before the fruit could be harvested. In the case of GDD, the heat requirements were 329 and 1246 for full bloom and fruit harvest, respectively. According to plant growth measurements, shoot growth lasted ≈7 months with a significant increase in the growth rate after fruit harvest reaching a maximum value in July. Trunk growth followed a similar annual pattern as that of the shoots but with its maximum rate occurring ≈30 days latter. Fruit growth, which lasted an average of 89 days from full bloom to harvesting, took place under mild climatic conditions (10 Feb. to 10 May) coinciding with only 30% of the total annual shoot length. This pattern of reproductive and vegetative growth pointed to the interest of redirecting regulated deficit irrigation practices in early-maturing cultivars toward postharvest water-saving strategies, but only to the extent that any limitation of shoot and trunk growth does not adversely affect the productivity of the following year.
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Giafi, C. F., and G. Rumsby. "Kinetic Analysis and Tissue Distribution of Human D-Glycerate Dehydrogenase/Glyoxylate Reductase and its Relevance to the Diagnosis of Primary Hyperoxaluria Type 2." Annals of Clinical Biochemistry: International Journal of Laboratory Medicine 35, no. 1 (January 1998): 104–9. http://dx.doi.org/10.1177/000456329803500114.
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The enzyme D-glycerate dehydrogenase (D-GDH; EC 1.1.1.29), which is also believed to have glyoxylate reductase (GR; EC 1.1.1.26/79) activity, plays a role in serine catabolism and glyoxylate metabolism and deficiency of this enzyme is believed to be the cause of primary hyperoxaluria type 2 (PH2). The pH optima and kinetic parameters of D-GDH and GR in human liver have been determined and assays developed for their measurement. Maximal activities were observed at pH 6.0, 8.0 and 7.6 for the D-GDH forward, D-GDH reverse and GR reactions, respectively. The apparent Km values for the substrates in these reactions were as follows: D-GDH forward reaction, 0.5 mmol/L hydroxypyruvate and 0.08 mmol/L NADPH; D-GDH reverse reaction, 20 mmol/L D-glycerate and 0.03 mmol/L NADP and for the GR reaction 1.25 mmol/L glyoxylate and 0.33 mmol/L NADPH. The forward D-GDH and GR assays were adopted for routine use, the low activity of the reverse D-GDH reaction being of little use for routine analyses. D-GDH and GR activity in 13 normal livers ranged from 350–940 nmol per min per mg protein (median 547) and 129–209 nmol per min per mg protein (median 145), respectively. D-GDH activity in kidney, lymphocytes and fibroblasts fell within the range of values seen in the liver but GR activity was approximately 30% in the kidney and barely detectable in lymphocytes and fibroblasts. Analysis of liver and lymphocyte samples from patients with PH2 showed that GR activity was either very low or undetectable while D-GDH activity was reduced in liver but within the normal range in lymphocytes. These results suggest that there is more than one enzyme with D-GDH activity in human tissues but only one of these has significant GR activity. We conclude that a definitive diagnosis of PH2 requires measurement of GR and D-GDH in a liver biopsy.
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Paulton, Joshua, Amanjot Gill, and Joelle Prevost. "GUT-DIRECTED SELF-HYPNOSIS FOR INFLAMMATORY BOWEL DISEASE PROTOCOL: COMPLIMENTARY PSYCHOTHERAPY FOR REMISSION AUGMENTATION, IBS-LIKE SYMPTOMS, AND SURGERY RECOVERY." Inflammatory Bowel Diseases 27, Supplement_1 (January 1, 2021): S53. http://dx.doi.org/10.1093/ibd/izaa347.126.
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Abstract Background Gut-directed hypnosis (GDH) is a complimentary therapy for Inflammatory Bowel Disease (IBD), that can be learnt by patients to practice self-hypnosis. GDH in IBD has augmented remission and improved inflammation. GDH has a history of successful use for Irritable Bowel Syndrome (IBS). In IBD it may also improve IBS-like symptoms in remission and recovery from surgery. GDH is suitable for youth and adult IBD patients. In hypnosis, a relaxed state is inducted then suggestions to subconscious mind processes are made. In IBD, the mechanism of action of GDH is unknown but may influence the disease stress response. Aims Aims are the development of a GDH self-hypnosis protocol for IBD, with appropriate target symptoms. Patients first learn to practice with a clinician, then as complimentary psychotherapy for remission augmentation, IBS-like symptoms, and surgery recovery. Methods GDH is practiced first with a clinician, and then by patients as self-hypnosis (table 1). Patients receive psycho-education on GDH for IBD. Next, appropriate treatment goals are made, based on target symptoms. Relaxation techniques induce patient to a deeply relaxed state. Therapeutic suggestions specific to patient goals are given: verbal suggestions, visualizations, and post-hypnotic suggestions. Suggestions can focus on having a healthy digestive system, inflammation and symptoms reduction, and achievement and sustainment of remission. Patients emerge from hypnosis, are debriefed, and encouraged to practice ongoing self-hypnosis. Results In IBD, GDH self-hypnosis can be learnt from clinicians and practiced by patients as a complimentary therapy. Patients’ achievement and sustainment of remission, with clinical markers of inflammation can be monitored. Patients can monitor subjective improvement of IBS-like symptoms and post surgery, recovery progress can be monitored. Conclusions GDH has a history of use for IBS. In IBD, it has been shown to modulate remission, and may improve IBS-like symptoms, and in surgery recovery. The mechanism of action of GDH in IBD may influence the disease stress response. Clinicians trained in GDH are limited currently. Patients may learn GDH self- hypnosis to as a complimentary psychotherapy.
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Paulton, J. P., J. Prevost, and A. K. Gill. "A179 GUT-DIRECTED SELF-HYPNOSIS FOR INFLAMMATORY BOWEL DISEASE PROTOCOL: COMPLIMENTARY PSYCHOTHERAPY FOR REMISSION AUGMENTATION, IBS-LIKE SYMPTOMS, AND SURGERY RECOVERY." Journal of the Canadian Association of Gastroenterology 4, Supplement_1 (March 1, 2021): 192–94. http://dx.doi.org/10.1093/jcag/gwab002.177.
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Abstract Background Gut-directed hypnosis (GDH) is a complimentary therapy for Inflammatory Bowel Disease (IBD), that can be learnt by patients to practice self-hypnosis. GDH in IBD has augmented remission and improved inflammation. GDH has a history of successful use for Irritable Bowel Syndrome (IBS). In IBD it may also improve IBS-like symptoms in remission and recovery from surgery. GDH is suitable for youth and adult IBD patients. In hypnosis, a relaxed state is inducted then suggestions to subconscious mind processes are made. In IBD, the mechanism of action of GDH is unknown but may influence the disease stress response. Aims Aims are the development of a GDH self-hypnosis protocol for IBD, with appropriate target symptoms. Patients first learn to practice with a clinician, then as complimentary psychotherapy for remission augmentation, IBS-like symptoms, and surgery recovery. Methods GDH is practiced first with a clinician, and then by patients as self-hypnosis (table 1). Patients receive psycho-education on GDH for IBD. Next, appropriate treatment goals are made, based on target symptoms. Relaxation techniques induce patient to a deeply relaxed state. Therapeutic suggestions specific to patient goals are given: verbal suggestions, visualizations, and post-hypnotic suggestions. Suggestions can focus on having a healthy digestive system, inflammation and symptoms reduction, and achievement and sustainment of remission. Patients emerge from hypnosis, are debriefed, and encouraged to practice ongoing self-hypnosis. Results In IBD, GDH self-hypnosis can be learnt from clinicians and practiced by patients as a complimentary therapy. Patients’ achievement and sustainment of remission, with clinical markers of inflammation can be monitored. Patients can monitor subjective improvement of IBS-like symptoms and post surgery, recovery progress can be monitored. Conclusions GDH has a history of use for IBS. In IBD, it has been shown to modulate remission, and may improve IBS-like symptoms, and in surgery recovery. The mechanism of action of GDH in IBD may influence the disease stress response. Clinicians trained in GDH are limited currently. Patients may learn GDH self-hypnosis as a complimentary psychotherapy. Funding Agencies None
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Wilson, David F., Abigail T. J. Cember, and Franz M. Matschinsky. "Glutamate dehydrogenase: role in regulating metabolism and insulin release in pancreatic β-cells." Journal of Applied Physiology 125, no. 2 (August 1, 2018): 419–28. http://dx.doi.org/10.1152/japplphysiol.01077.2017.
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Regulation of insulin release and glucose homeostasis by pancreatic β-cells is dependent on the metabolism of glucose by glucokinase (GK) and the influence of that activity on oxidative phosphorylation. Genetic alterations that result in hyperactivity of mitochondrial glutamate dehydrogenase (GDH-1) can cause hypoglycemia-hyperammonemia following high protein meals, but the role of GDH-1 remains poorly understood. GDH-1 activity is strongly inhibited by GTP, to near zero in the absence of ADP, and cooperatively activated ( n = 2.3) by ADP. The dissociation constant for ADP is near 200 µM in vivo, but leucine and its nonmetabolized analog 2-amino-2-norbornane-carboxylic acid (BCH) can activate GDH-1 by increasing the affinity for ADP. Under physiological conditions, as [ADP] increases GDH-1 activity remains very low until ~35 µM (threshold) and then increases rapidly. A model for GDH-1 and its regulation has been combined with a previously published model for glucose sensing that coupled GK activity and oxidative phosphorylation. The combined model (GK-GDH-core) shows that GK activity determines the energy state ([ATP]/[ADP][Pi]) in β-cells for glucose concentrations > 5 mM ([ADP] < 35 µM). As glucose falls < 5 mM the [ADP]-dependent increase in GDH-1 activity prevents [ADP] from rising above ~70 µM. Thus, GDH-1 dynamically buffers β-cell energy metabolism during hypoglycemia, maintaining the energy state and the basal rate of insulin release. GDH-1 hyperactivity suppresses the normal increase in [ADP] in hypoglycemia. This leads to hypoglycemia following a high protein meal by increasing the basal rate of insulin release (β-cells) and decreasing glucagon release (α-cells). NEW & NOTEWORTHY A model of β-cell metabolism and regulation of insulin release is presented. The model integrates regulation of oxidative phosphorylation, glucokinase (GK), and glutamate dehydrogenase (GDH-1). GDH-1 is near equilibrium under physiological conditions, but the activity is inhibited by GTP. In hypoglycemia, however, GK activity is low and [ADP], a potent activator of GDH-1, increases. Reducing equivalents from GDH dynamically buffers the intramitochondrial [NADH]/[NAD+], and thereby the energy state, preventing hypoglycemia-induced substrate deprivation.
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Andreas, Plaitakis, Flessas Panayiotis, B. Natsiou Anastasia, and P. Shashidharan. "Glutamate Dehydrogenase Deficiency in Cerebellar Degenerations: Clinical, Biochemical and Molecular Genetic Aspects." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 20, S3 (May 1993): S109—S116. http://dx.doi.org/10.1017/s0317167100048617.
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ABSTRACT:Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is significantly reduced in patients with heterogenous neurological disorders characterized by multiple system atrophy (MSA) and predominant involvement of the cerebellum and its connections. In human brain, GDH exists in multiple isoforms differing in their isoelectric point and molecular mass. These are differentially reduced in quantity and altered in catalytic activity in patients with clinically distinct forms of MSA, thus suggesting that these GDH isoproteins are under different genetic control. Dysregulation of glutamate metabolism occurs in patients with GDH deficiency and is thought to mediate the disease’s neurodegeneration via neuroexcitotoxic mechanisms. This possibility is supported by additional data showing that glutamate binding sites are significantly decreased in cerebellar tissue obtained at autopsy from MSA patients. At the molecular biological level, several cDNAs specific for human GDH have been isolated recently and cloned. Northern blot analysis of various human tissues, including brain, has revealed the presence of multiple GDH-specific mRNAs. In addition, multiple GDH-specific genes are present in humans and these data are consistent with the possibility that the various GDH isoproteins are encoded by different genes. These advances have laid the groundwork for characterizing the human GDH genes and their products in health and disease.
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Geerts, W. J., M. Verburg, A. Jonker, A. T. Das, L. Boon, R. Charles, W. H. Lamers, and C. J. Van Noorden. "Gender-dependent regulation of glutamate dehydrogenase expression in periportal and pericentral zones of rat liver lobules." Journal of Histochemistry & Cytochemistry 44, no. 10 (October 1996): 1153–59. http://dx.doi.org/10.1177/44.10.8813080.
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We studied the level(s) at which glutamate dehydrogenase (GDH; EC 1.4.1.2) expression is regulated in the livers of fed male and female rats. The cellular content of GDH mRNA, protein, and enzyme activity was determined quantitatively using image analysis for measurement of the absorbance in consecutive serial sections that were processed for in situ hybridization, immunohistochemistry, and enzyme histochemistry. In both males and females, GDH protein and activity patterns were similar, with pericentral values being twice as high as periportal values. GDH mRNA distribution patterns in female liver lobules reflected those of GDH protein and activity, but GDH mRNA distribution patterns in male rat livers were found to be homogeneous owing to a more than twofold lower cellular mRNA content in pericentral zones than in female rats. We conclude that gender affects GDH expression selectively in pericentral zones at posttranscriptional and pretranslational levels.
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Zhao, Yanjie, Jie Gao, Shengzhong Su, Xiaohui Shan, Shipeng Li, Hongkui Liu, Yaping Yuan, and He Li. "Regulation of the activity of maize glutamate dehydrogenase by ammonium and potassium." Bioscience, Biotechnology, and Biochemistry 85, no. 2 (December 31, 2020): 262–71. http://dx.doi.org/10.1093/bbb/zbaa020.
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ABSTRACT Glutamate dehydrogenase (GDH) is an important enzyme in ammonium metabolism, the activity of which is regulated by multiple factors. In this study, we investigate the effects of ammonium and potassium on the activity of maize GDH. Our results show that both ammonium and potassium play multiple roles in regulating the activity of maize GDH, with the specific roles depending on the concentration of potassium. Together with the structural information of GDH, we propose models for the substrate inhibition of ammonium, and the elimination of substrate inhibition by potassium. These models are supported by the analysis of statistic thermodynamics. We also analyze the binding sites of ammonium and potassium on maize GDH, and the conformational changes of maize GDH. The findings provide insight into the regulation of maize GDH activity by ammonium and potassium and reveal the importance of the dose and ratio of nitrogen and potassium in crop cultivation.
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Han, Qingye, Weili Gong, Zhenyu Zhang, Lushan Wang, Binglian Wang, Lei Cai, Qingjun Meng, et al. "Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay." International Journal of Molecular Sciences 22, no. 11 (May 24, 2021): 5529. http://dx.doi.org/10.3390/ijms22115529.
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The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10−2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10−2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.
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Haferkamp, Patrick, Simone Kutschki, Jenny Treichel, Hatim Hemeda, Karsten Sewczyk, Daniel Hoffmann, Melanie Zaparty, and Bettina Siebers. "An additional glucose dehydrogenase from Sulfolobus solfataricus: fine-tuning of sugar degradation?" Biochemical Society Transactions 39, no. 1 (January 19, 2011): 77–81. http://dx.doi.org/10.1042/bst0390077.
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Within the SulfoSYS (Sulfolobus Systems Biology) project, the effect of temperature on a metabolic network is investigated at the systems level. Sulfolobus solfataricus utilizes an unusual branched ED (Entner–Doudoroff) pathway for sugar degradation that is promiscuous for glucose and galactose. In the course of metabolic pathway reconstruction, a glucose dehydrogenase isoenzyme (GDH-2, SSO3204) was identified. GDH-2 exhibits high similarity to the previously characterized GDH-1 (SSO3003, 61% amino acid identity), but possesses different enzymatic properties, particularly regarding substrate specificity and catalytic efficiency. In contrast with GDH-1, which exhibits broad substrate specificity for C5 and C6 sugars, GDH-2 is absolutely specific for glucose. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, whereas GDH-1 might have a dominant role in galactose degradation via the same pathway as well as in different sugar-degradation pathways.
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Basith, Shaherin, Balachandran Manavalan, Tae Hwan Shin, and Gwang Lee. "Mapping the Intramolecular Communications among Different Glutamate Dehydrogenase States Using Molecular Dynamics." Biomolecules 11, no. 6 (May 27, 2021): 798. http://dx.doi.org/10.3390/biom11060798.
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Glutamate dehydrogenase (GDH) is a ubiquitous enzyme that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate. It acts as an important branch-point enzyme between carbon and nitrogen metabolisms. Due to the multifaceted roles of GDH in cancer, hyperinsulinism/hyperammonemia, and central nervous system development and pathologies, tight control of its activity is necessitated. To date, several GDH structures have been solved in its closed form; however, intrinsic structural information in its open and apo forms are still deficient. Moreover, the allosteric communications and conformational changes taking place in the three different GDH states are not well studied. To mitigate these drawbacks, we applied unbiased molecular dynamic simulations (MD) and network analysis to three different GDH states i.e., apo, active, and inactive forms, for investigating their modulatory mechanisms. In this paper, based on MD and network analysis, crucial residues important for signal transduction, conformational changes, and maps of information flow among the different GDH states were elucidated. Moreover, with the recent findings of allosteric modulators, an allosteric wiring illustration of GDH intramolecular signal transductions would be of paramount importance to obtain the process of this enzyme regulation. The structural insights gained from this study will pave way for large-scale screening of GDH regulators and could support researchers in the design and development of new and potent GDH ligands.
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Rasgado, Lourdes A. Vega, Guillermo Ceballos Reyes, and Fernando Vega Díaz. "Modulation of brain glutamate dehydrogenase as a tool for controlling seizures." Acta Pharmaceutica 65, no. 4 (December 1, 2015): 443–52. http://dx.doi.org/10.1515/acph-2015-0033.
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Abstract Glutamate (Glu) is a major excitatory neurotransmitter involved in epilepsy. Glu is synthesized by glutamate dehydrogenase (GDH, E.C. 1.4.1.3) and dysfunction of the enzymatic activity of GDH is associated with brain pathologies. The main goal of this work is to establish the role of GDH in the effects of antiepileptic drugs (AEDs) such as valproate (VALP), diazepam (DIAZ) and diphenylhydantoin (DPH) and its repercussions on oxygen consumption. Oxidative deamination of Glu and reductive amination of aketoglutarate (αK) in mice brain were investigated. Our results show that AEDs decrease GDH activity and oxygen consumption in vitro. In ex vivo experiments, AEDs increased GDH activity but decreased oxygen consumption during Glu oxidative deamination. VALP and DPH reversed the increase in reductive amination of αK caused by the chemoconvulsant pentylenetetrazol. These results suggest that AEDs act by modulating brain GDH activity, which in turn decreased oxygen consumption. GDH represents an important regulation point of neuronal excitability, and modulation of its activity represents a potential target for metabolic treatment of epilepsy and for the development of new AEDs.
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Wright, P. A., R. K. Packer, A. Garcia-Perez, and M. A. Knepper. "Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats." American Journal of Physiology-Renal Physiology 262, no. 6 (June 1, 1992): F999—F1006. http://dx.doi.org/10.1152/ajprenal.1992.262.6.f999.
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To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.
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Maly, I. P., and D. Sasse. "Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver." Journal of Histochemistry & Cytochemistry 39, no. 8 (August 1991): 1121–24. http://dx.doi.org/10.1177/39.8.1856459.
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In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.
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Vetterli, Laurène, Stefania Carobbio, Shirin Pournourmohammadi, Rafael Martin-del-Rio, Dorte M. Skytt, Helle S. Waagepetersen, Jorge Tamarit-Rodriguez, and Pierre Maechler. "Delineation of glutamate pathways and secretory responses in pancreatic islets with β-cell–specific abrogation of the glutamate dehydrogenase." Molecular Biology of the Cell 23, no. 19 (October 2012): 3851–62. http://dx.doi.org/10.1091/mbc.e11-08-0676.
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In pancreatic β-cells, glutamate dehydrogenase (GDH) modulates insulin secretion, although its function regarding specific secretagogues is unclear. This study investigated the role of GDH using a β-cell–specific GDH knockout mouse model, called βGlud1−/−. The absence of GDH in islets isolated from βGlud1–/– mice resulted in abrogation of insulin release evoked by glutamine combined with 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid or l-leucine. Reintroduction of GDH in βGlud1–/– islets fully restored the secretory response. Regarding glucose stimulation, insulin secretion in islets isolated from βGlud1–/– mice exhibited half of the response measured in control islets. The amplifying pathway, tested at stimulatory glucose concentrations in the presence of KCl and diazoxide, was markedly inhibited in βGlud1–/– islets. On glucose stimulation, net synthesis of glutamate from α-ketoglutarate was impaired in GDH-deficient islets. Accordingly, glucose-induced elevation of glutamate levels observed in control islets was absent in βGlud1–/– islets. Parallel biochemical pathways, namely alanine and aspartate aminotransferases, could not compensate for the lack of GDH. However, the secretory response to glucose was fully restored by the provision of cellular glutamate when βGlud1–/– islets were exposed to dimethyl glutamate. This shows that permissive levels of glutamate are required for the full development of glucose-stimulated insulin secretion and that GDH plays an indispensable role in this process.
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Girinathan, Brintha Prasummanna, Sterling E. Braun, and Revathi Govind. "Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2." Microbiology 160, no. 1 (January 1, 2014): 47–55. http://dx.doi.org/10.1099/mic.0.071365-0.
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Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile‐associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity.
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FANG, Jie, Betty Y. L. HSU, Courtney M. MacMULLEN, Mortimer PONCZ, Thomas J. SMITH, and Charles A. STANLEY. "Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations." Biochemical Journal 363, no. 1 (March 22, 2002): 81–87. http://dx.doi.org/10.1042/bj3630081.
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Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED50, 210μM) and S448P (ED50, 3.1μM) human GDH mutants compared with the wild-type human GDH (ED50, 42nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH.
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Engel, Paul C. "Making biochemistry count: life among the amino acid dehydrogenases." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 425–29. http://dx.doi.org/10.1042/bst0390425.
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The guiding principle of the IAS Medal Lecture and of the research it covered was that searching mathematical analysis, depending on good measurements, must underpin sound biochemical conclusions. This was illustrated through various experiences with the amino acid dehydrogenases. Topics covered in the present article include: (i) the place of kinetic measurement in assessing the metabolic role of GDH (glutamate dehydrogenase); (ii) the discovery of complex regulatory behaviour in mammalian GDH, involving negative co-operativity in coenzyme binding; (iii) an X-ray structure solution for a bacterial GDH providing insight into catalysis; (iv) almost total positive co-operativity in glutamate binding to clostridial GDH; (v) unexpected outcomes with mutations at the catalytic aspartate site in GDH; (vi) reactive cysteine as a counting tool in the construction of hybrid oligomers to probe the basis of allosteric interaction; (vii) tryptophan-to-phenylalanine mutations in analysis of allosteric conformational change; (viii) site-directed mutagenesis to alter substrate specificity in GDH and PheDH (phenylalanine dehydrogenase); and (ix) varying strengths of binding of the ‘wrong’ enantiomer in engineered mutant enzymes and implications for resolution of racemates.
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Piatti, Gabriella, Marco Bruzzone, Vincenzo Fontana, and Marcello Ceppi. "Analysis of Routine and Integrative Data from Clostridioides difficile Infection Diagnosis and the Consequent Observations." Open Microbiology Journal 13, no. 1 (December 31, 2019): 343–49. http://dx.doi.org/10.2174/1874285801913010343.
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Background: Clostridioides difficile Infection (CDI) is an acute disease that needs a fast proper treatment. Unfortunately, the diagnosis, and above all the understanding of the results, remain arduous. Objective: This study analyzed routine and integrative results of all fecal samples from patients over time. Our aim was to understand the dynamics of CDI infection and the meaning of “difficult to interpret” results, to make physicians better understand the various tools they can use. Methods: We evaluated routine results obtained from 815 diarrheal stools with Enzyme Immunoassay (EIA) that detects C. difficile Glutamate Dehydrogenase (GDH) antigen and toxin B. We also reanalyzed a part of samples using integrative tests: a Real-time polymerase chain reaction (RT-PCR) for C. difficile toxin B gene (tcdB) and the automated immunoassay VIDAS C. difficile system for GDH and toxins A/B. Results: EIA GDH positivity increased through multiple testing over time, with a P value <0.001, depicting a sort of bacterial growth curve. Eighty-five percent of GDH positive/toxin B negative, i.e., discrepant, samples PCR were tcdB positive, 61.5% of discrepant tcdB positive samples were VIDAS toxins A/B positive, and 44.4% of GDH EIA negative stools were VIDAS GDH positive. Conclusion: The results confirmed the low sensitivity of the EIA system for C. difficile GDH and toxins, questioned the use of the latter for concluding any CDI diagnostic algorithm, and led us to indicate the algorithm beginning with tcdB molecular research, and continuing in positive cases with VIDAS CD GDH method, as the most effective for CDI.
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Lee, Jinhee, Atsuro Tatsumi, Kaori Tsukakoshi, Ellie D. Wilson, Koichi Abe, Koji Sode, and Kazunori Ikebukuro. "Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF." Sensors 20, no. 14 (July 11, 2020): 3878. http://dx.doi.org/10.3390/s20143878.
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Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.
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Costa, A. Lombardi, M. Cherubini, F. D'Auria, W. Giannotti, and A. Moskalev. "Thermal-Hydraulic Analysis of Coolant Flow Decrease in Fuel Channels of Smolensk-3 RBMK during GDH Blockage Event." Science and Technology of Nuclear Installations 2007 (2007): 1–7. http://dx.doi.org/10.1155/2007/87834.
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One of the transients that have received considerable attention in the safety evaluation of RBMK reactors is the partial break of a group distribution header (GDH). The coolant flow rate blockage in one GDH might lead to excessive heat-up of the pressure tubes and can result in multiple fuel channels (FC) ruptures. In this work, the GDH flow blockage transient has been studied considering the Smolensk-3 RBMK NPP (nuclear power plant). In the RBMK, each GDH distributes coolant to 40–43 FC. To investigate the behavior of each FC belonging to the damaged GDH and to have a more realistic trend, one (affected) GDH has been schematized with its forty-two FC, one by one. The calculations were performed using the 0-D NK (neutron kinetic) model of the RELAP5-3.3 stand-alone code. The results show that, during the event, the mass flow rate is disturbed differently according to the power distribution established for each FC in the schematization. The start time of the oscillations in mass flow rate depends strongly on the attributed power to each FC. It was also observed that, during the event, the fuel channels at higher thermal power values tend to undergo first cladding rupture leaving the reactor to scram and safeguarding all the other FCs connected to the affected GDH.
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Kaiser, S., J. J. Hwang, H. Smith, C. Banner, T. C. Welbourne, and N. P. Curthoys. "Effect of altered acid-base balance and of various agonists on levels of renal glutamate dehydrogenase mRNA." American Journal of Physiology-Renal Physiology 262, no. 3 (March 1, 1992): F507—F512. http://dx.doi.org/10.1152/ajprenal.1992.262.3.f507.
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Rat kidney contains 3.5-kb and 2.8-kb mRNAs that encode for glutamate dehydrogenase (GDH). The levels of both mRNAs are increased gradually after onset of chronic metabolic acidosis and reach a maximum induction of 2.5-fold after 7 days. In contrast, during recovery from chronic acidosis, the levels of the GDH mRNAs are returned to normal within 1 day. The development of an acute metabolic acidosis causes a more rapid induction of GDH mRNA. This increase occurs after a 7-h lag and plateaus after 18 h at a level that is threefold greater than normal. A very similar profile was observed after the transfer of LLC-PK-F+ cells from normal medium to an acidic medium containing 10 mM bicarbonate and adjusted to pH 6.9. However, the transfer of cells from acidic to normal medium caused an immediate and rapid [half-life (t) = 1 h] decrease in GDH mRNA. The apparent half-lives of GDH mRNA were measured by treating cells grown in normal (t = 4 h) and acidic media (t = 12 h) with actinomycin D. Thus, increased stability may account for the induction of GDH mRNA that occurs during growth in response to acidosis. The levels of GDH mRNA are independently affected by changes in medium pH or bicarbonate concentration. The levels of GDH mRNA are also increased by treating cells with adenosine 3',5'-cyclic monophosphate, epinephrine, triiodothyronine, or retinoic acid, whereas treatment with angiotensin II, vasopressin, phorbol 12-myristate 13-acetate, or cycloheximide did not produce an increase. The inductive effect of dexamethasone, which is observed in vivo, is not reproduced in the LLC-PK-F+ cells.
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Noor, Shahid, and Narayan S. Punekar. "Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface." Microbiology 151, no. 5 (May 1, 2005): 1409–19. http://dx.doi.org/10.1099/mic.0.27751-0.
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NADP-dependent glutamate dehydrogenase (NADP-GDH) mediates fungal ammonium assimilation through reductive synthesis of glutamate from 2-oxoglutarate. By virtue of its position at the interface of carbon and nitrogen metabolism, biosynthetic NADP-GDH is a potential candidate for metabolic control. In order to facilitate characterization, a new and effective dye-affinity method was devised to purify NADP-GDH from two aspergilli, Aspergillus niger and Aspergillus nidulans. The A. niger NADP-GDH was characterized at length and its kinetic interaction constants with glutamate (K m 34·7 mM) and ammonium (K m 1·05 mM; K i 0·4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective K i values of 6·9, 9·2 and 202·0 μM. The A. niger enzyme showed allosteric properties and a sigmoid response (n H=2·5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH from Aspergillus awamori, A. nidulans and Aspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli.
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Gunawardena, Indunil. "Effectiveness of the geriatric day hospital – a realist review." Reviews in Clinical Gerontology 21, no. 3 (February 22, 2011): 267–69. http://dx.doi.org/10.1017/s0959259811000050.
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SummaryThis research paper is a realist review on the effectiveness of medical care for older people in the geriatric day hospital (GDH), based on trial data from a Cochrane review published in 2008. The Cochrane review indicated no overall difference between GDH care and alternative services. However, health care management and policy interventions are quite complex and methodologically more diverse than clinical treatments. Hence a ‘realist review’ is a more suitable explanatory analysis, aimed at discerning what works for whom, in what circumstances, in what respect, and how. This realist review on GDH care provides an explanatory analysis and has aimed to identify where GDH care is and is not effective.
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Srivastava, D. K., P. Smolen, G. F. Betts, T. Fukushima, H. O. Spivey, and S. A. Bernhard. "Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?" Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6464–68. http://dx.doi.org/10.1073/pnas.86.17.6464.
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Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that NADH is transferred directly between the sites of alpha-GDH and LDH.
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Rajas, F., and B. Rousset. "A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity." Biochemical Journal 295, no. 2 (October 15, 1993): 447–55. http://dx.doi.org/10.1042/bj2950447.
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We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme.
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Thatcher, Bradley J., and Kenneth B. Storey. "Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: Evidence for two distinct enzyme forms." Biochemistry and Cell Biology 79, no. 1 (January 1, 2001): 11–19. http://dx.doi.org/10.1139/o00-086.
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Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37°C body temperature) and hibernating (torpid, 5°C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5 ± 2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335 ± 5 kDa for euthermic and 320 ± 5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and α-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5°C). For example, the Km for α-ketoglutarate of euthermic GDH was higher at 5°C (3.66 ± 0.34 mM) than at 37°C (0.10 ± 0.01 mM), whereas hibernator GDH had a higher affinity for α-ketoglutarate at 5°C (Km was 0.98 ± 0.08 mM at 37°C and 0.43 ± 0.02 mM at 5°C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37°C and weakest with hibernator GDH at 5°C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.Key words: mammalian hibernation, amino acid metabolism, temperature-dependent enzyme kinetics, Spermophilus richardsonii.
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Treberg, Jason R., Kathy A. Clow, Katie A. Greene, Margaret E. Brosnan, and John T. Brosnan. "Systemic activation of glutamate dehydrogenase increases renal ammoniagenesis: implications for the hyperinsulinism/hyperammonemia syndrome." American Journal of Physiology-Endocrinology and Metabolism 298, no. 6 (June 2010): E1219—E1225. http://dx.doi.org/10.1152/ajpendo.00028.2010.
Full textAbstract:
The hyperinsulism/hyperammonemia (HI/HA) syndrome is caused by glutamate dehydrogenase (GDH) gain-of-function mutations that reduce the inhibition by GTP, consequently increasing the activity of GDH in vivo. The source of the hyperammonemia in the HI/HA syndrome remains unclear. We examined the effect of systemic activation of GDH on ammonia metabolism in the rat. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) is a nonmetabolizable analog of the natural GDH allosteric activator leucine. A dose of 100 μmol BCH/100 g rat resulted in a mild systemic hyperammonemia. Using arterial-venous (A-V) differences, we exclude the liver, intestine, and skeletal muscle as major contributors to this BCH-induced hyperammonemia. However, renal ammonia output increased, as demonstrated by an increase in A-V difference for ammonia across the kidney in BCH-treated animals. Isolated renal cortical tubules incubated with BCH increased the rate of ammoniagenesis from glutamine by 40%. The flux through GDH increased more than twofold when BCH was added to renal mitochondria respiring on glutamine. The flux through glutaminase was not affected by BCH, whereas glutamate-oxaloacetate transaminase flux decreased when normalized to glutaminase flux. These data show that increased renal ammoniagenesis due to activation of GDH can explain the BCH-induced hyperammonemia. These results are discussed in relation to the organ source of the ammonia in the HI/HA syndrome as well as the role of GDH in regulating renal ammoniagenesis.
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Crilly, Richard G., Sonya Lylwynec, Marita Kloseck, Jan M. Smith, Tyler Olsen, Bill Gold, and Shelley Masse. "Patient Outcomes after Discharge from a Geriatric Day Hospital." Canadian Journal on Aging / La Revue canadienne du vieillissement 24, no. 3 (2005): 305–9. http://dx.doi.org/10.1353/cja.2005.0076.
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ABSTRACTEvidence suggests that frailer older patients benefit from a continuum of care rather than the admit/discharge model of our health system. This study examined patient outcomes after discharge from a geriatric day hospital (GDH) to determine what proportion continues to do well, what proportion declines, how the two groups differ, and if factors predictive of deterioration can be identified. Using telephone survey and Goal Attainment Scaling methodologies, the goals of 151 patients discharged from a GDH between 6 and 18 months previously were examined to determine whether GDH achievements were maintained or lost. All but 5 patients improved between GDH admission and discharge; after discharge, 39 per cent deteriorated. The need for more support in the community was predictive of deterioration, probably reflecting patient frailty. Number of medical diagnoses or medications were not predictive. Frailer older patients tend not to maintain goals achieved in a GDH after discharge and may benefit from ongoing maintenance.
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Floré, Katelijne M. J., and Joris R. Delanghe. "Analytical Interferences in Point-of-Care Testing Glucometers by Icodextrin and its Metabolites: An Overview." Peritoneal Dialysis International: Journal of the International Society for Peritoneal Dialysis 29, no. 4 (July 2009): 377–83. http://dx.doi.org/10.1177/089686080902900403.
Full textAbstract:
Current point-of-care testing (POCT) glucometers are based on various test principles. Two major method groups dominate the market: glucose oxidase-based systems and glucose dehydrogenase-based systems using pyrroloquinoline quinone (GDH-PQQ) as a cofactor. The GDH-PQQ-based glucometers are replacing the older glucose oxidase-based systems because of their lower sensitivity for oxygen. On the other hand, the GDH-PQQ test method results in falsely elevated blood glucose levels in peritoneal dialysis patients receiving solutions containing icodextrin ( e.g., Extraneal; Baxter, Brussels, Belgium). Icodextrin is metabolized in the systemic circulation into different glucose polymers, but mainly maltose, which interferes with the GDH-PQQ-based method. Clinicians should be aware of this analytical interference. The POCT glucometers based on the GDH-PQQ method should preferably not be used in this high-risk population and POCT glucose results inconsistent with clinical suspicion of hypoglycemic coma should be retested with another testing system.
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Helbing, K., G. Anton, M. Fausten, D. Menze, T. Michel, A. Nagel, D. Ryckbosch, T. Speckner, R. Van de Vyver, and G. Zeitler. "The GDH-Detector." Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 484, no. 1-3 (May 2002): 129–39. http://dx.doi.org/10.1016/s0168-9002(01)01988-x.
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Vega Rasgado, Lourdes A., Guillermo Ceballos Reyes, and Fernando Vega-Díaz. "Anticonvulsant Drugs, Brain Glutamate Dehydrogenase Activity and Oxygen Consumption." ISRN Pharmacology 2012 (March 8, 2012): 1–8. http://dx.doi.org/10.5402/2012/295853.
Full textAbstract:
Glutamate dehydrogenase (GDH, E.C. 1.4.1.3.) is a key enzyme for the biosynthesis and modulation of glutamate (GLU) metabolism and an indirect γ-aminobutyric acid (GABA) source, here we studied the effect of anticonvulsants such as pyridoxal phosphate (PPAL), aminooxyacetic acid (AAOA), and hydroxylamine (OHAMINE) on GDH activity in mouse brain. Moreover, since GLU is a glucogenic molecule and anoxia is a primary cause of convulsions, we explore the effect of these drugs on oxygen consumption. Experiments were performed in vitro as well as in vivo for both oxidative deamination of GLU and reductive amination of α-ketoglutarate (αK). Results in vitro showed that PPAL decreased oxidative deamination of GLU and oxygen consumption, whereas AAOA and OHAMINE inhibited GDH activity competitively and also inhibited oxygen consumption when αK reductive amination was carried out. In contrast, results showed that in vivo, all anticonvulsants enhanced GLU utilization by GDH and also decreased oxygen consumption. Together, results suggest that GDH activity has repercussions on oxygen consumption, which may indicate that the enzyme activity is highly regulated by energy requirements for metabolic activity. Besides, GDH may participate in regulation of GLU and, indirectly GABA levels, hence in neuronal excitability, becoming a key enzyme in seizures mechanism.
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Aghajanian, S. A., S. R. Martin, and P. C. Engel. "Urea-induced inactivation and denaturation of clostridial glutamate dehydrogenase: the absence of stable dimeric or trimeric intermediates." Biochemical Journal 311, no. 3 (November 1, 1995): 905–10. http://dx.doi.org/10.1042/bj3110905.
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Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5′-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective. This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate. Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.
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Gaspar, Carlos, Jonás I. Silva-Marrero, María C. Salgado, Isabel V. Baanante, and Isidoro Metón. "Role of upstream stimulatory factor 2 in glutamate dehydrogenase gene transcription." Journal of Molecular Endocrinology 60, no. 3 (April 2018): 247–59. http://dx.doi.org/10.1530/jme-17-0142.
Full textAbstract:
Glutamate dehydrogenase (Gdh) plays a central role in ammonia detoxification by catalysing reversible oxidative deamination of l-glutamate into α-ketoglutarate using NAD+ or NADP+ as cofactor. To gain insight into transcriptional regulation of glud, the gene that codes for Gdh, we isolated and characterised the 5′ flanking region of glud from gilthead sea bream (Sparus aurata). In addition, tissue distribution, the effect of starvation as well as short- and long-term refeeding on Gdh mRNA levels in the liver of S. aurata were also addressed. 5′-Deletion analysis of glud promoter in transiently transfected HepG2 cells, electrophoretic mobility shift assays, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis allowed us to identify upstream stimulatory factor 2 (Usf2) as a novel factor involved in the transcriptional regulation of glud. Analysis of tissue distribution of Gdh and Usf2 mRNA levels by reverse transcriptase-coupled quantitative real-time PCR (RT-qPCR) showed that Gdh is mainly expressed in the liver of S. aurata, while Usf2 displayed ubiquitous distribution. RT-qPCR and ChIP assays revealed that long-term starvation down-regulated the hepatic expression of Gdh and Usf2 to similar levels and reduced Usf2 binding to glud promoter, while refeeding resulted in a slow but gradual restoration of both Gdh and Usf2 mRNA abundance. Herein, we demonstrate that Usf2 transactivates S. aurata glud by binding to an E-box located in the proximal region of glud promoter. In addition, our findings provide evidence for a new regulatory mechanism involving Usf2 as a key factor in the nutritional regulation of glud transcription in the fish liver.
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Anno, Takatoshi, Shunsuke Uehara, Hideki Katagiri, Yasuharu Ohta, Kohei Ueda, Hiroyuki Mizuguchi, Yoshinori Moriyama, Yoshitomo Oka, and Yukio Tanizawa. "Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation." American Journal of Physiology-Endocrinology and Metabolism 286, no. 2 (February 2004): E280—E285. http://dx.doi.org/10.1152/ajpendo.00380.2003.
Full textAbstract:
Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to α-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native β-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to α-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.
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Jaqueti Aroca, Jerónimo, Laura M. Molina Esteban, Isabel García-Arata, Jesús García-Martínez, Isabel Cano De Torres, and Santiago Prieto Menchero. "Significance of a polymerase chain reaction method in the detection of Clostridioides difficile." Revista Española de Quimioterapia 34, no. 2 (February 19, 2021): 141–44. http://dx.doi.org/10.37201/req/010.2020.
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Objectives. Clostridioides difficile (CD) is the most common cause of nosocomial diarrhea. Detection of CD toxin in patients’ faecal samples is the traditional rapid method for the diagnosis of CD infection. Various testing algorithms have been proposed: an initial screening test using a rapid test, and a confirmatory test (cytotoxicity neutralization assay, toxigenic culture, nucleic acid amplification test) for discordant results. The aim of this study was to evaluate the effectiveness of a two-step algorithm using an immunochromatographic test followed of a polymerase chain reaction (PCR). Material and methods. The specimens have been tested according to the following schedule: 1) Step one: All samples were tested for detection of glutamate dehydrogenase antigen (GDH) and toxin A/B using the C. diff QUIK CHEK Complete test. All GDH and toxins positive results were considered CD positives; 2) Step two: When the results were discrepant (only GDH+ or toxins+), the samples were confirmed using the PCR test BD MAX Cdiff. All PCR positive results were considered CD positives. Results. A total of 2,138 specimens were initially tested. 139 were positive for GDH and toxins. 160 discrepant results (148 GDH+ and 12 toxins+) were tested by PCR, 117 were positive (107/148 GDH+ and 10/12 toxins+). Conclusions. The implementation of a PCR method showed an increase de 117 positive results (73.1% of discrepant). Considering the sensitivity of C.diff QUIK CHEK (instructions of manufacturer), the GDH discrepant results may be false negatives, y the samples PCR and toxins positives may be real positives results.
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Hashim, Shehab, Dong-Hyeon Kwon, Ahmed Abdelal, and Chung-Dar Lu. "The Arginine Regulatory Protein Mediates Repression by Arginine of the Operons Encoding Glutamate Synthase and Anabolic Glutamate Dehydrogenase in Pseudomonas aeruginosa." Journal of Bacteriology 186, no. 12 (June 15, 2004): 3848–54. http://dx.doi.org/10.1128/jb.186.12.3848-3854.2004.
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ABSTRACT The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD+-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in α-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of β-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.