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Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal ammonia-generating component. We show that alkalization is dependent on the SPS-sensor-regulated transcription factor STP2 and the proline-responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2-/- strain. Alkalization is dependent on mitochondrial activity and in wild-type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co-culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal-induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal-associated processes are more critical in promoting dysbiosis and virulent fungal growth.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Nitrate and ammonium are the main nitrogen sources in agricultural soils. In the last decade, ammonium (NH4+), a double-sided metabolite, has attracted considerable attention by researchers. Its ubiquitous presence in plant metabolism and its metabolic energy economy for being assimilated contrast with its toxicity when present in high amounts in the external medium. Plant species can adopt different strategies to maintain NH4+ homeostasis, as the maximization of its compartmentalization and assimilation in organic compounds, primarily as amino acids and proteins. In the present study, we report an integrative metabolic response to ammonium nutrition of seven plant species, belonging to four different families: Gramineae (ryegrass, wheat, Brachypodium distachyon), Leguminosae (clover), Solanaceae (tomato), and Brassicaceae (oilseed rape, Arabidopsis thaliana). We use principal component analysis (PCA) and correlations among metabolic and biochemical data from 40 experimental conditions to understand the whole-plant response. The nature of main amino acids is analyzed among species, under the hypothesis that those Asn-accumulating species will show a better response to ammonium nutrition. Given the provision of carbon (C) skeletons is crucial for promotion of the nitrogen assimilation, the role of different anaplerotic enzymes is discussed in relation to ammonium nutrition at a whole-plant level. Among these enzymes, isocitrate dehydrogenase (ICDH) shows to be a good candidate to increase nitrogen assimilation in plants. Overall, metabolic adaptation of different carbon anaplerotic activities is linked with the preference to synthesize Asn or Gln in their organs. Lastly, glutamate dehydrogenase (GDH) reveals as an important enzyme to surpass C limitation during ammonium assimilation in roots, with a disparate collaboration of glutamine synthetase (GS).

For millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anticancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing antiviral responses that eliminate tumors through mechanisms not completely understood. Here, we find that massive binding of epigenetically activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2 family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics. SIGNIFICANCE: The state of viral mimicry induced by epigenetic therapies in cancer cells remodels mitochondrial metabolism and drives caspase-independent tumor cell death, which sensitizes to BCL2 inhibitor drugs. This novel mechanism underlies clinical efficacy of hypomethylating agents and venetoclax in acute myeloid leukemia, suggesting similar combination therapies for other incurable cancers.

Ionotropic glutamate delta receptors do not bind glutamate and do not generate ionic current, resulting in difficulty in studying the function and trafficking of these receptors. Here, we utilize chimeric constructs, in which the ligand-binding domain of GluD1 is replaced by that of GluK1, to examine its synaptic trafficking and plasticity. GluD1 trafficked to the synapse, but was incapable of expressing long-term potentiation (LTP). The C-terminal domain (CT) of GluD1 has a classic PDZ-binding motif, which is critical for the synaptic trafficking of other glutamate receptors, but we found that its binding to PSD-95 was very weak, and deleting the PDZ-binding motif failed to alter synaptic trafficking. However, deletion of the entire CT abolished synaptic trafficking, but not surface expression. We found that mutation of threonine (T) T923 to an alanine disrupted synaptic trafficking. Therefore, GluD1 receptors have strikingly different trafficking mechanisms compared with AMPARs. These results highlight the diversity of ionotropic glutamate receptor trafficking rules at a single type of synapse. Since this receptor is genetically associated with schizophrenia, our findings may provide an important clue to understand schizophrenia.

QC 20220301

Transdeamination seems to be an important alternative energy-intensive gluconeogenesis mechanism that generates glucose from non-carbohydrate sources during pupal-adult metamorphosis in Bombyx mori. Studies on four transdeamination parameters, viz., free amino acids (FAA), aspartate aminotransferase (AAT), alanine aminotransferase (AlAT) and glutamate dehydrogenase (GDH) in the fat body and haemolymph of silkworm have indicated that transdeamination shows stage-specific, region-specific and sex-specific variations during metamorphosis. Region-specific growth trends indicate that the transamination reaction, mediated by AAT and AlAT is faster in the abdominal fat body (AFB) and relatively slower in the thoracic fat body (TFB) with concomitant lower FAA levels in the former and higher levels in the latter. Stage-specific growth trends reflect that the energy mobilization through transdeamination assumes greater significance in the early pupal, late pupal and adult stages rather than the mid-pupal stage. Sex-wise trends in FAA levels indicate that the rate of amino acid utilization is significantly faster in females compared to that in males. Further, the growth trends in the levels of GDH vis-à-vis aminotransferases signify that the energy demands of male sex expressions are met largely through enhanced levels of GDH and that the alpha ketoglutarate generated in transamination reaction is used as a substrate for sperm production, sperm motility and successful mating that stimulates fecundity and productivity of in the mulberry silkworm. The study clearly demonstrates that gluconeogenesis through transdeamination supplements the energy requirements of silkworm metamorphosis and that it is facilitated by disintegrating tissues predominantly from the pupal abdominal segments.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long-Evans (L-E). Time course and dose-response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L-E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L-E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L-E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L-E rats.

Ammonium nutrition often represents an important growth-limiting stress in plants. Some of the symptoms that plants present under ammonium nutrition have been associated with pH deregulation, in fact external medium pH control is known to improve plants ammonium tolerance. However, the way plant cell metabolism adjusts to these changes is not completely understood. Thus, in this work we focused on how Arabidopsis thaliana shoot and root respond to different nutritional regimes by varying the nitrogen source (NO3 and NH4), concentration (2 and 10 mM) and pH of the external medium (5.7 and 6.7) to gain a deeper understanding of cell metabolic adaptation upon altering these environmental factors. The results obtained evidence changes in the response of ammonium assimilation machinery and of the anaplerotic enzymes associated to Tricarboxylic Acids (TCA) cycle in function of the plant organ, the nitrogen source and the degree of ammonium stress. A greater stress severity at pH 5.7 was related to NH4 accumulation; this could not be circumvented in spite of the stimulation of glutamine synthetase, glutamate dehydrogenase, and TCA cycle anaplerotic enzymes. Moreover, this study suggests specific functions for different gin and gdh isoforms based on the nutritional regime. Overall, NH4 accumulation triggering ammonium stress appears to bear no relation to nitrogen assimilation impairment.

Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician.

Warming has been shown to cause soil carbon (C) loss in northern grasslands owing to accelerated microbial decomposition that offsets increased grass productivity. Yet, a multi-decadal survey indicated that the surface soil C stock in Tibetan alpine grasslands remained relatively stable. To investigate this inconsistency, we analyzed the feedback responses of soil microbial communities to simulated warming by soil transplant in Tibetan grasslands. Whereas microbial functional diversity decreased in response to warming, microbial community structure did not correlate with changes in temperature. The relative abundance of catabolic genes associated with nitrogen (N) and C cycling decreased with warming, most notably in genes encoding enzymes associated with more recalcitrant C substrates. By contrast, genes associated with C fixation increased in relative abundance. The relative abundance of genes associated with urease, glutamate dehydrogenase and ammonia monoxygenase (ureC, gdh and amoA) were significantly correlated with N2O efflux. These results suggest that unlike arid/semiarid grasslands, Tibetan grasslands maintain negative feedback mechanisms that preserve terrestrial C and N pools. To examine whether these trends were applicable to the whole plateau, we included these measurements in a model and verified that topsoil C stocks remained relatively stable. Thus, by establishing linkages between microbial metabolic potential and soil biogeochemical processes, we conclude that long-term C loss in Tibetan grasslands is ameliorated by a reduction in microbial decomposition of recalcitrant C substrates.

It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4+ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4+when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4+. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4+ when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD (H)-dependent GDH activity

Cultured astrocytes treated with siRNA to knock down glutamate dehydrogenase (GDH) were used to investigate whether this enzyme is important for the utilization of glutamate as an energy substrate. By incubation of these cells in media containing different concentrations of glutamate (range 100-500 µM) in the presence or in the absence of glucose, the metabolism of these substrates was studied by using tritiated glutamate or 2-deoxyglucose as tracers. In addition, the cellular contents of glutamate and ATP were determined. The astrocytes were able to maintain physiological levels of ATP regardless of the expression level of GDH and the incubation condition, indicating a high degree of flexibility with regard to regulatory mechanisms involved in maintaining an adequate energy level in the cells. Glutamate uptake was found to be increased in these cells when exposed to increasing levels of extracellular glutamate independently of the GDH expression level. Moreover, increased intracellular glutamate content was observed in the GDH-deficient cells after a 2-hr incubation in the presence of 100 µM glutamate. It is significant that GDH-deficient cells exhibited an increased utilization of glucose in the presence of 250 and 500 µM glutamate, monitored as an increase in the accumulation of tritiated 2-deoxyglucose-6-phosphate. These findings underscore the importance of the expression level of GDH for the ability to utilize glutamate as an energy source fueling its own energy-requiring uptake.

Introduction: Eight Giardia duodenalis genotypes (A-H) have been described to date. Genotypes A and B have been isolated from humans and a wide range of mammals; however, genotypes C-H have shown greater host specificity.Objective: Identifying G. duodenalis genotypes from cysts in faeces obtained from children attending the Instituto Colombiano de Bienestar Familiar (ICBF) day care centres and from dogs in Ibagué by PCR-RFLP targeting both the β-giardin and glutamate dehydrogenase genes.Materials and methods: Cysts from G. duodenalis positive samples were concentrated, DNA was extracted and the β-giardin and glutamate dehydrogenase genes were analysed by PCR-RFLP. The MHOM/CO/04/G40 strain was used as positive control (this was obtained from the Grupo de Parasitología at the Instituto Nacional de Salud).Results: Of the total human samples, 11/23 (48%) were genotyped as A and 12/23 (52%) as B; PCR-RFLP revealed that four canine samples were genotypes C and D, these being host-specific.Conclusions: Only genotypes associated with human infection (AII, BIII and BIV) were found in the children and host-specific genotypes were observed in canines (C and D). No interaction could be established between animal and human transmission cycles due to the small canine sample size and as the former did not come into contact with children attending ICBF day-care centres., Introducción. Se han descrito ocho genotipos de Giardia duodenalis, del A al H. Los genotipos A y B se han aislado de humanos y de una gran variedad de mamíferos; sin embargo, los genotipos del C al H han mostrado mayor especificidad de huésped.Objetivo. Identificar los genotipos de G. duodenalis a partir de quistes obtenidos en heces de niños de las guarderías del Instituto Colombiano de Bienestar Familiar (ICBF) y de perros en Ibagué, mediante PCR-RFLP de los genes de la beta giardina y la glutamato deshidrogenasa.Materiales y métodos. Los quistes de las muestras positivas para G. duodenalis fueron sometidos a concentración; se extrajo su ADN y se efectuó el análisis de PCR-RFLP de los genes de la beta giardina y de la glutamato deshidrogenasa. Como control positivo se utilizó la cepa MHOM/CO/04/G40 procedente del Grupo de Parasitología del Instituto Nacional de Salud.Resultados. De las muestras tomadas de niños, 11/23 (48 %) correspondieron al genotipo A y, 12/23 (52 %), al genotipo B. Cuatro muestras de perros presentaron los genotipos C y D, específicos de este huésped.Conclusiones. En los niños solamente se encontraron los genotipos asociados a infecciones humanas (AII, BIII y BIV) y en los perros, los genotipos específicos para este huésped (C y D). Debido al reducido tamaño de las muestras analizadas provenientes de perros, y dado que estos no estuvieron en contacto con los niños de las guarderías del ICBF, no fue posible determinar una interacción entre el ciclo de transmisión de los humanos y el de los animales.

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.

This is the first study to characterise proline, arginine and lysine metabolism in homogenates of L3 and adult Haemonchus contortus and Teladorsagia circumcincta. The properties of glutamate dehydrogenase (GDH), glutamate synthase and the GABA shunt were also compared in the two species. The kinetic properties of 26 enzymes were determined. The gene encoding T. circumcincta GDH was sequenced and recombinant TcGDH expressed and biochemically characterised. The ornithine-glutamate-proline pathway was fully functional. The mammalian α-AAA (saccharopine) and pipecolate pathways of lysine catabolism, but not the bacterial enzymes lysine dehydrogenase and decarboxylase, were present in adult worms. The pipecolate pathway was incomplete in L3 of both species, as Pip2CR activity was undetectable. Unusually, lysine ketoglutarate reductase and saccharopine dehydrogenase, Δ1-pyrroline-5-carboxylate synthase and reductase were able to use both cofactors. The glutamine synthetase-glutamate synthase pathway of ammonia incorporation into glutamate was present, except in L3 H. contortus. T. circumcincta GDH was cloned, purified and characterised and the predicted protein sequence was very similar to H. contortus GDH. T. circumcincta recombinant and H. contortus homogenate GDH were both dual co-factor specific, although the latter had 50% greater activity with NAD+/H as cofactor. GDH activity was inhibited by GTP and stimulated by ADP whereas ATP either inhibited or stimulated depending on the concentration and direction of the reaction. The GABA shunt enzymes glutamate decarboxylase and succinic semialdehyde dehydrogenase was not detected in homogenates of whole L3 or adult H. contortus or T. circumcincta. Neither parasite had a full functional ornithine urea cycle, nor appeared to use bacterial pathways to covert arginine to ornithine. NOS were demonstrated histochemically in nerves of adult H. contortus, but was undetectable in homogenates of both species. There was species varia

Glutamátdehydrogenáza (GLDH) je enzym patřící do třídy oxidoreduktáz.Uplatnění nachází pri metabolismu dusíku, kde katalyzuje oxidační deaminaci glutamátu a také hraje důležitou roli v udržení energetiské homeostázy. Aktivita enzymu výrazně stoupá v krvi při nekróze jaterních buněk.Nejčastější metodou stanovení GLDH je spektrofotometrie, kdy je detekován pokles absorbance způsobený oxidací NADH při 340 nm., Glutamate dehydrogenase (GLDH) belongs to the oxidoreductase class of enzymes.The aim of our study was to optimize the method for determionation of glutamate dehydrogenase activity, and to find out the optimal conditions for enzyme stability., Katedra biologických a biochemických věd, 1. Prezentace výsledků diplomové práce 2. Diskuze k posudkům vedoucího a oponenta diplomové práce 3. Diplomantka zodpověděla všechny dotazy a připomínky k DP.

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture. The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp (L. vannamei) were investigated. This involved an examination of growth performance, glutamate dehydrogenase (GDH) and Na+-K+ ATPase mRNA expression,, and GDH activity in muscles and gills. Three experimental diets were formulated, containing 25%, 40%, and 50% dietary protein, and fed to the shrimp at a salinity of 25. After 20 days, no significant difference was observed in weight gain, though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels. Subsequently, shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5, respectively, and sampled at weeks 1 and 2. Shrimp fed with 40% protein at 25 in salinity (optimal conditions) were used as a control. Regardless of the salinities, shrimp fed with 50% dietary protein had significantly higher growth performance than other diets; no significant differences were found in comparison with the control. Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks. Ambient salinity change also stimulated the hepatosomatic index, which increased in the first week and then recovered to a relatively normal level, as in the control, after 2 weeks. These findings indicate that in white shrimp, the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism. Increased dietary protein level could improve the osmoreaulation capacity of L. vannamei with more energy resources allocated to GDH activity and expression.

Die Resistenzentwicklung des bedeutendsten humanpathogenen Malariaerregers, Plasmodium falciparum, gegen vorhandene Wirkstoffe, nimmt seit den 60er Jahren stetig zu und bedingt seit den 90er Jahren nahezu 50 % der Gesamtsterblichkeitsrate innerhalb der afrikanischen Bevölkerung. Aus diesem Grund ist es von besonderer Bedeutung, neue Zielmoleküle zu identifizieren und darauf basierend effizienter wirkende Antimalariamedikamente zu entwickeln. Ziel der vorliegenden Arbeit war es daher, die Glutamat-Dehydrogenasen 2 und 3 aus Plasmodium falciparum detailliert auf molekularer Ebene zu charakterisieren, um letztlich ihre Eignung als sogenannte drug targets zu validieren. Der Schwerpunkt der vorliegenden Arbeit lag auf der biochemischen und strukturellen Charakterisierung des zweiten Glutamat-Dehydrogenase Isoenzyms aus Plasmodium falciparum. Die Klonierung unterschiedlich langer PfGDH2 Konstrukte, die heterologe Überexpression dieser in E. coli Zellen und eine anschließende Separation der Proteine aus dem Bakterienlysat erbrachte letztlich ein um 49 Aminosäuren verkürztes, katalytisch aktives Enzym. Anschließend wurde die PfGDH2 (hemm-)kinetisch charakterisiert. Weiterhin wurde über proteinkristallographische Methoden die dreidimensionale Kristallstruktur des rekombinanten PfGDH2 Proteins bestimmt. Eine Überlagerung dieser Struktur mit denen bereits charakterisierter GDHs, zeigte eine große Übereinstimmung der Sekundärstrukturmerkmale mit Glutamat-Dehydrogenasen der S_50I Familie unterschiedlicher Organismen. Die an der Koordination der Substrate beteiligten Aminosäurereste sind auch in diesem Protein stark konserviert. Eine Analyse der Monomer-Monomer-Interaktionen ergab jedoch deutliche Unterschiede im Vergleich zum bereits charakterisierten ersten GDH-Isoenzym aus Plasmodium falciparum. Untersuchungen zur Lokalisation aller drei GDH-Proteine des Parasiten über Fusionsproteine mit dem green fluorescent protein (GFP) wurden durchgeführt, wodurch Hinweise darauf, in welc, The development of resistance in the most important human malaria parasite, Plasmodium falciparum, against currently available drugs has been steadily increasing since the 1960s and has accounted for nearly 50 % of all deaths in the African population since the 1990s. For this reason it is of great importance to identify new target molecules and to develop novel and efficient antimalarials. The main goal of the present work was to characterize the glutamate dehydrogenases 2 and 3 from Plasmodium falciparum on a molecular level, in order to validate their suitability as drug targets. The focus was on the biochemical and structural characterization of the second glutamate dehydrogenase isoenzyme from Plasmodium falciparum. Cloning PfGDH2 constructs of varying lengths, heterologously overexpressing them and then separating the proteins via metal chelate affinity chromatography finally resulted in a catalytically active enzyme (truncated by 49 amino acids). Subsequently, the PfGDH2 protein was kinetically characterized. Furthermore the three-dimensional crystal structure of the apo form of the hexameric PfGDH2 protein was determined and examined at a molecular level. Superimposing this structure onto those of other GDH crystal structures showed great similarity in the secondary structure features, which correspond to the generally known structure of glutamate dehydrogenases in the S_50I family of various organisms. Also for PfGDH2 all amino acids that participate in the coordination of the substrates are highly conserved. In contrast to this however, the analysis of the monomer-monomer interactions showed significant differences in comparison to the first GDH isoenzyme of the parasite. Furthermore, studies to localize all three GDH proteins within the parasites via fusion proteins with the green fluorescent protein (GFP) were conducted. In silico analyses including virtual docking for structure-based drug discovery were conducted for both PfGDH1 and PfGDH2 as well as fo

Ammonia, being highly toxic and readily soluble in water, is excreted by aquatic animals like fishes. However, scarcity of water in the surrounding medium, may force the fishes to convert ammonia into urea. In the present study, the possible role of ureogenesis to avoid accumulation of toxic ammonia under water-restricted condition was tested in Labeo rohita. Blood urea and ammonia were estimated in the blood of the fishes and glutamate dehydrogenase acitivity was measured in the hepatic tissue. From the present study, however, it is found that the relationship between blood ammonia and blood urea in Labeo rohita and the possible role of glutamate dehydrogenase is not so pronounced in the experimental fish species.

Introduction : Congenital hyperinsulinism and hyperammonemia (CHH) is caused by gain of function of glutamate dehydrogenase (GDH). The genetic abnormalities are known to be located in three specific regions on the GDH protein. We describe here three different missense mutations identified in five new Japanese patients with CHH. And to study the genotype-phenotype correlations in patients with GLUD1 mutations, we analyzed previously reported Japanese cases. / Methods : An Epstein-Barr virus-transformed lymphoblastoid cell line was established from the 5 patients and control subjects, and was used for enzymatic and molecular analyses....

A great deal of research in cheese technology is devoted towards the manipulation of glycolysis, lipolysis and proteolysis in order to accelerate ripening times or to improve flavour. There is increasing evidence that certain yeast species contribute to flavour and texture development during ripening of certain cheeses, including Cheddar cheese. The addition of yeast cultures as adjunct cultures may accelerate ripening, or lead to a faster development of flavour and taste. This is brought about by an increase in primary proteolysis of the caseins as well as the break down of amino acids into flavour compounds. The respective enzymes involved seem to be proteases with plasmin or plasmin activator activity, and glutamate dehydrogenase. This research reports on the inclusion of yeasts as adjuncts in the processing of Cheddar cheese. Yeasts that expressed both enzyme activities were added as single adjuncts, while combinations of yeasts with singular enzyme activity were employed. Changes during ripening were monitored by sensory and biochemical analyses. The latter includes proteolysis, lipolysis and enzyme activity.

Congenital hyperinsulinism (CHI), characterized by profound hypoglycaemia related to inappropriate insulin secretion, may be associated histologically with either diffuse insulin hypersecretion or focal adenomatous hyperplasia, which share a similar clinical presentation, but result from different molecular mechanisms. Whereas diffuse CHI is of autosomal recessive, or less frequently of autosomal dominant, inheritance, focal CHI is sporadic. The most common mechanism underlying CHI is dysfunction of the pancreatic ATP-sensitive potassium channel (K(+)(ATP)). The two subunits of the K(+)(ATP) channel are encoded by the sulfonylurea receptor gene (SUR1 or ABCC8) and the inward-rectifying potassium channel gene (KIR6.2 or KCNJ11), both located in the 11p15.1 region. Germ-line, paternally inherited, mutations of the SUR1 or KIR6.2 genes, together with somatic maternal haplo-insufficiency for 11p15.5, were shown to result in focal CHI. Diffuse CHI results from germ-line mutations in the SUR1 or KIR6.2 genes, but also from mutations in several other genes, namely glutamate dehydrogenase (with associated hyperammonaemia), glucokinase, short-chain L-3-hydroxyacyl-CoA dehydrogenase, and insulin receptor gene. Hyperinsulinaemic hypoglycaemia may be observed in several overlapping syndromes, such as Beckwith-Wiedemann syndrome (BWS), Perlman syndrome, and, more rarely, Sotos syndrome. Mosaic genome-wide paternal isodisomy has recently been reported in patients with clinical signs of BWS and CHI. The primary causes of CHI are genetically heterogeneous and have not yet been completely unveiled. However, secondary causes of hyperinsulinism have to be considered such as fatty acid oxidation deficiency, congenital disorders of glycosylation and factitious hypoglycaemia secondary to Munchausen by proxy syndrome.

A bienzyme sensor for the amperometric determination of ammonium (NH4+) was constructed by immobilizing glutamate oxidase (GXD, E.C. 1.4.3.11) and glutamate dehydrogenase (GIDH. E.C. 1.4.1.2) on a Clark-type oxygen electrode, The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. GIDH consumes ammonium for specific amination of 2-oxoglutarate in the presence of NADH. GXD consumes dissolved oxygen for the oxidative deamination of glutamate produced by GIDH. Dissolved oxygen acts as an essential material for GXD during its enzymatic reactions. Therefore, a signal detected as the maximum rate of the consumption of dissolved oxygen by GXD, was observed in the measurement of ammonium. A Teflon membrane was used to fabricate the biosensor in order to avoid interferences from real samples. The biosensor has a fast response (2 s) and short recovery time (2 min), The total test time for a measurement by using this ammonium biosensor (4 min) was faster than using a commercial ammonium testing kit (up to 20 min). The biosensor has a linear range between 10 and 300 μ m ammonium, with a detection limit of 2.06 μ M. A good agreement (R-2 = 0,9984) with a commercial ammonium testing kit was obtained in the measurement of wastewater sample. © 2004 Elsevier B.V. All rights reserved.

Glucose and other sugars, such as galactose or maltose, are able to cause carbon catabolite repression in Saccharomyces cerevisiae. Although glycolytic intermediates have been suggested as signal for repression, no evidence for such a control mechanism is available. The establishment of a correlation between levels of intracellular metabolites and the extent of catabolite repression may facilitate the identification of potential signal molecules in the process. To set a framework for such a study, the repression produced by xylose, glycerol and dihydroxyacetone upon genes belonging to different repressible circuits was tested, using an engineered strain of S. cerevisiae able to metabolize xylose. Xylose decreased the derepression of various enzymes in the presence of ethanol by at least 10-fold; the corresponding mRNAs were not detected in these conditions. Xylose also impaired the derepression of galactokinase and invertase. Glycerol and dihydroxyacetone decreased 2- to 3-fold the derepression observed in ethanol or galactose but did not affect invertase derepression. For yeast cells grown in media with different carbon sources, no correlation was found between repression of fructose-1,6-bisphosphatase and intracellular levels of glucose 6-phosphate or fructose 1,6-bisphosphate.

Persistent hyperinsulinism is the most common cause of recurrent hypoglycemia in infancy because of inappropriate oversecretion of insulin by the pancreas. Pancreatic lesions can be either focal or diffuse, and they have distinct molecular bases. We have studied the facial features in 17 unrelated patients presenting with neonatal (n = 8) or infancy-onset (n = 9) hyperinsulinism. Hyperinsulinism was related to focal adenomatous hyperplasia (n = 7), diffuse hyperinsulinism (n = 5), non-operated hyperinsulinism (n = 2), and hyperinsulinism with hyperammonemia (n = 3). SUR1 or Kir6.2 mutations were found in six of seven focal adenomatous hyperplasia and three of five diffuse hyperinsulinism. A loss of the maternal allele from chromosome 11p15 in the lesion was found in all focal adenomatous hyperplasia. GLUD1 mutations were found in all patients with hyperammonemia. Large birth weight (mean > 3,800 g) was consistently observed (11/17) but protruding tongue, exomphalos, or visceromegaly were never noted and Wiedemann-Beckwith syndrome could always be ruled out. All patients presented with high forehead, small nasal tip, and short columella giving the impression that the nose is large and bulbous, smooth philtrum, and thin upper lip. A square appearance to the face was more obvious in younger patients. These specific facial features, observed in patients with hyperinsulinism of various molecular mechanisms, could be the consequence of fetal intoxication by insulin. However, to date, facial anomalies have not been noted in infants of diabetic mothers and inversely, malformations that are commonly reported in infants of diabetic mothers were not present in our hyperinsulinemic patients.

Glutamate dehydrogenases (GDHs) from fresh-water and marine hyperthermophilic Archaea were compared with respect to their responses to different salt concentrations. A gene encoding GDH from the terrestrial hyperthermophilic archaeon Thermococcus waiotapuensis (Twaio) was cloned, sequenced, and expressed at a high level in Escherichia coli. The deduced amino acid sequence, which consists of 418 amino acid residues, revealed a high degree of similarity with GDHs from related marine strains such as Thermococcus litoralis (Tl) and Pyrococcus furiosus (Pfu). Recombinant Twaio GDH was purified 27-fold to homogeneity. The enzyme is hexameric with a molecular weight of 259,000. The effects of several salts (KCl, CaCl, MgSO4), temperature, and pH on enzyme activity were determined and compared in three hyperthermophilic GDHs, including T. waiotapuensis, and GDHs from two marine species, T. litoralis and P. furiosus. Kinetic studies suggested a biosynthetic role for the nicotinamide adenine dinucleotide phosphate- (NADP-) specific Twaio GDH in the cell. Interestingly, Twaio GDH revealed no salt responses, whereas the two marine GDHs showed substantial enhancement of activity as well as thermostability at increasing salt concentrations. Because electrostatic interactions between charged amino acid residues are thought to be a key feature of structural integrity and thermostability in hyperthermophilic GDHs, salt availability and its effects on marine enzymes could partially explain a higher thermal stability in marine species than in phyletically related fresh-water species.

A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (-61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2-2 phenotype.

Few studies examine ammonia and amino acid metabolism in response to endurance training Trained humans generally experience less increase in plasma ammonia during either prolonged or intense exercise. This is probably a reflection of reduced ammonia production and release from the active muscle; it could be a reflection of decreased AMP deaminase activity, decreased glutamate dehydrogenase activity, and/or increased alanine and glutamine formation. Little is known regarding the associated enzyme systems in humans, but in experiments with animal models, aerobic training decreases AMP deaminase and increases the enzymes of amino acid transamination and oxidation.

The effect of five Azotobacter chroococcum strains and nitrogen content in nutrient media on callus growth of two Beta vulgaris L. cultivars were investigated, as well as the activity of nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in inoculated callus tissue. On medium with full nitrogen content (1 N) the inoculation with A. chroococcum strain A(2) resulted in the highest calli mass, while strains A(8) and A(14) maximally increased NR activity. On media with 1/8 N the highest effect on calli growth, GS and GDH activity had the strain A(8). The strain A(2/1) significantly increased callus proliferation on medium without N. Asymbiotic association between sugar beet calli and Azotobacter depended on genotype/strain interaction and was realised in presence of different nitrogen levels.

The treatment of patients with hepatic failure by means of hybrid liver support devices using primary xenogeneic hepatocytes is currently hindered by the rapid loss of cell metabolic functions. Similarly to what happens with other mammalian cells, accumulation of catabolites in the neighborhood of cultured hepatocytes might significantly affect their viability and functions. In this paper, we investigated the effects of high concentrations of catabolites, such as ammonia and lactic acid, on the viability and functions of rat hepatocytes cultured on collagen coated Petri dishes. The effects on hepatocyte functions were established with respect to their ability to synthesize urea and to eliminate ammonia. Indeed, high catabolite concentrations effected both hepatocyte viability and functions. The number of viable hepatocytes decreased with increasing ammonia concentrations in the culture medium. High ammonia concentrations had also both an inhibitory and a toxic effect on hepatocyte functions. In fact, the hepatocytes synthesized urea and eliminated ammonia at rates that decreased with increasing ammonia concentrations. Similarly, high lactic acid concentrations were toxic to the cells and also inhibited their synthetic functions.

Young wheat (C3) and maize (C4) plants were exposed to near-ambient concentrations of ozone in open-top chambers in order to investigate the possible effects of ozone on nitrogen metabolism. Nitrogen was supplied to the plants by adding 15N-labelled tracer substances via the soil substrate. Enzyme activities (NADH nitrate reductase, nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase) and the incorporation of 15N were determined.The findings show that nitrogen metabolism was affected by O3, however, there were distinct differences between the two species. In plants treated with O3, NADH nitrate reductase activity in maize leaves was reduced, while NR activity in wheat leaves only slightly declined. Only minor changes were observed with respect to the activities of nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase.Feeding experiments using 15NO3 − showed that the incorporation of nitrate nitrogen in wheat plants exposed to ozone remains virtually unchanged, whereas in maize plants reduced incorporation rates were observed for nitrate nitrogen. The incorporation of ammonium nitrogen was distinctly increased in wheat and maize by the impact of ozone.When investigating pigment contents, reduced levels of chlorophyll a and b and carotenoids were observed, whereas the pigment content of wheat leaves remained unchanged. These results indicate that young maize plants are more susceptible than wheat plants to short-term ozone exposure.

SCOPUS: ar.j, info:eu-repo/semantics/published

The inferior colliculus of the genetically epilepsy-prone rat (GEPR) was examined at the ultrastructural level to determine if any abnormalities exist in the inferior colliculus of the GEPR as compared to the non-epileptic Sprague-Dawley rat. Both routine electron microscopic preparations and glutamate decarboxylase (GAD) and GABA immunocytochemical preparations were examined in the GEPR and compared to previous studies from this laboratory that described the normal ultrastructure of the Sprague-Dawley rat. Cell counts from 2 micron semi-thin sections confirmed our previous observations that showed a large, significant increase in the number of neurons in the inferior colliculus of the GEPR as compared to the Sprague-Dawley rat. Many of the small neurons in the inferior colliculus of the GEPR were found to be smaller than those in the inferior colliculus of the Sprague-Dawley rat. Moreover, the small neurons in the GEPR were frequently clumped in clusters of 3-5. Several ultrastructural abnormalities present in the inferior colliculus of the GEPR have been observed at epileptic foci or in brain regions along the pathway of seizure spread in other experimental models of epilepsy. These changes included the presence of dendrites which are almost completely devoid of organelles, hypertrophy of glial processes, and terminals that contain either swollen vesicles or very few vesicles. Other features that were frequently observed in the GEPR but were rarely found in preparations of Sprague-Dawley rats included an abundance of extra membranes, whorl bodies and multivesicular bodies within somata, dendrites and axons.(ABSTRACT TRUNCATED AT 250 WORDS)

An automatic continuous flow method is described for assay of plasmatic and urinary urea after hydrolysis by urease followed by NH4 assay with glutamic deshydrogenase in the same working solution. Precision and accuracy, comparison with chemical diacetylmonoxim method are studied. The interaction of endogenous NH4, the cost of the method are calculated.

The 6-phosphogluconate dehydrogenase (6-PGD), glucose-6-phosphate dehydrogenase (G-6-PD) and glutamate dehydrogenase (GDH) zymogram phenotypes of wheat, rye and their aneuploid derivates were determined. At least three genes involved in the production of 6-PGD isozymes were located on chromosome arms 4RL, 6RL and 2RL of ‘Imperial’ rye, ‘King II’ rye and ‘Dakold’ rye. Evidence was obtained that at least one gene located on chromosome arm 5RS controls G-6-PD isozyme activities in these varieties of rye and one gene involved in the production of GDH isozymes was located on chromosome arm 2RS of ‘Imperial’, ‘King II’ and ‘Dakold’ rye. The results indicate that the 6-PGD isozymes exist as dimers and monomers. No possible structure of the G-6-PD and GDH could be determined.