Glutamate-ammonia Ligase Research Articles

Gene Expression Changes in the Septum: Possible Implications for MicroRNAs in Sculpting the Maternal Brain

The transition from the non-maternal to the maternal state is characterized by a variety of CNS alterations that support the care of offspring. The septum (including lateral and medial portions) is a brain region previously linked to various emotional and motivational processes, including maternal care. In this study, we used microarrays (PLIER algorithm) to examine gene expression changes in the septum of postpartum mice and employed gene set enrichment analysis (GSEA) to identify possible regulators of altered gene expression. Genes of interest identified as differentially regulated with microarray analysis were validated with quantitative real-time PCR. We found that fatty acid binding protein 7 (Fabp7) and galanin (Gal) were downregulated, whereas insulin-like growth factor binding protein 3 (Igfbp3) was upregulated in postpartum mice compared to virgin females. These genes were previously found to be differentially regulated in other brain regions during lactation. We also identified altered expression of novel genes not previously linked to maternal behavior, but that could play a role in postpartum processes, including glutamate-ammonia ligase (Glul) and somatostatin receptor 1 (Sstr1) (both upregulated in postpartum). Genes implicated in metabolism, cell differentiation, or proliferation also exhibited altered expression. Unexpectedly, enrichment analysis revealed a high number of microRNAs, transcription factors, or conserved binding sites (177 with corrected P-value <0.05) that were significantly linked to maternal upregulated genes, while none were linked to downregulated genes. MicroRNAs have been linked to placenta and mammary gland development, but this is the first indication they may also play a key role in sculpting the maternal brain. Together, this study provides new insights into genes (along with possible mechanisms for their regulation) that are involved in septum-mediated adaptations during the postpartum period.

Abstract MP051: A Novel Genetic Locus for Coronary Heart Disease in Type 2 Diabetes

Genetic susceptibility to cardiovascular disease in type 2 diabetes is influenced by abnormal metabolic status in the affected diabetic patients; and the implicated genes may differ from those in the non-diabetic populations. We conducted 3-stage genome-wide association analysis to detect the genetic factors for coronary heart disease (CHD) in existing type 2 diabetes. We analyzed 2.4 million SNPs in 1,249 diabetic women from the Nurses’ Health Study (273 CHD cases and 976 controls) and 984 diabetic men from the Health Professionals’ Follow-up Study (319 CHD cases and 665 controls), all of European ancestry. The first-stage replication was performed in Joslin Heart Study (420 CHD cases and 431 controls); and the second-stage replication was performed in Gargano Heart Study (314 CHD cases and 384 controls), and Catanzaro study (114 CHD cases and 215 controls). All the replication samples were of European ancestry and also affected by type 2 diabetes. We observed associations of genome-wide significance (the best p=2.04 * 10 -8 ) between CHD and SNPs on chromosome 1, between genes ZNF648 ( zinc finger protein 648; 1q25 .3) and GLUL (glutamate-ammonia ligase; 1q31). eQTL analysis indicates suggestive association between the associated SNPs and a potential CHD gene PPAP2B on chromosome 1 with marginal significance (p=1 * 10 -5 ). The identified SNP showed a significant association with the plasma levels of vascular cell adhesion molecule 1 ( VCAM1 ) in patients with type 2 diabetes but not in non-diabetic healthy controls. Our findings indicate that specific genetic predisposition may determine cardiovascular risk in patients with type 2 diabetes. The findings may inform the development of novel therapies for reduction of cardiovascular complications in diabetes.

Cerebral gene expression and neurobehavioural development after perinatal exposure to an environmentally relevant polybrominated diphenylether (BDE47)

Nutrients in seafood are known to be beneficial for brain development. Effects of maternal exposure to 2,2′,4,4′ tetrabromo diphenylether (BDE47) was investigated, alongside the potential ameliorating impact of seafood nutrients, through assessment of neurobehaviour and gene expression in brain and liver. Developing mice were exposed during gestation and lactation via dams dosed through casein- or salmon-based feed, spiked with BDE47. Two concentrations were used: a low level (6 μg/kg feed) representing an environmentally realistic concentration and a high level (1,900 μg/kg feed) representing a BDE47 intake much higher than expected from frequents consumption of contaminated seafood. Experimental groups were similar with respect to reproductive success, growth and physical development. Minor, transient changes in neurobehavioural metrics were observed in groups given the highest dose of BDE47. No significant differences in behaviour or development were seen on postnatal day 18 among maternally exposed offspring. Cerebral gene expression investigated by microarray analyses and validated by RT-qPCR showed low fold changes for all genes, despite dose-dependent accumulation of BDE47 in brain tissue. The gene for glutamate ammonia ligase was upregulated compared to control in the casein-based high BDE47diet, suggesting potential impacts on downstream synaptic transmission. The study supported a previously observed regulation of Igfbp2 in brain with BDE47 exposure. Genes for hepatic metabolic enzymes were not influenced by BDE47. Potential neurotoxic effects and neurobehavioural aberrations after perinatal exposure to high levels of BDE47 were not readily observed in mice pups with the present experimental exposure regimes and methods of analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10565-011-9192-8) contains supplementary material, which is available to authorized users.

Molecular Reproduction &amp; Development Volume 78, Issue 2 cover image

Molecular Reproduction and DevelopmentVolume 78, Issue 2 p. C1-C1 Cover ImageFree Access Molecular Reproduction & Development Volume 78, Issue 2 cover image First published: 17 February 2011 https://doi.org/10.1002/mrd.21294AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Exposure to metals, such as nickel, can contribute to male subfertility, but the mechanisms involved are poorly understood. Glutamate-ammonia ligase (GLUL) was identified by Sun et al. (in this issue) as a prominent testicular nickel-binding protein whose activity is inhibited by nickel. Here, GLUL (green) is found in interstitial cells of the seminiferous tubules. Section was counterstained for nuclear DNA (DAPI, blue) and sperm (acetylated tubulin in tails, red). Volume78, Issue2February 2011Pages C1-C1 RelatedInformation

Binding of nickel to testicular glutamate-ammonia ligase inhibits its enzymatic activity

Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate-ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure.

Microarray analysis of the effect of dexamethasone on murine cochlear explants

Conclusions: The microarray analysis identified 39 genes up- or down-regulated by dexamethasone in the cultured tissue of mice cochlea. Of the eight genes most highly affected, several are suggested to have protective effects in the traumatized inner ear (Fkbp5, Glucocorticoid-induced leucine zipper (Gilz), glutathione peroxidase 3) and for others, a plausible mechanism of action can be offered (claudin 10, glutamate-ammonia ligase). The present data may support the use of dexamethasone to treat acute sensorineural hearing loss. It is warrantable to test these results in the in vivo cochlea. Objectives: To identify genes whose expression is markedly up- or down-regulated by dexamethasone in the cochlear tissue. Methods: Murine cochlear tissue was cultured with or without dexamethasone for 48 h in vitro. The gene expression profiles were compared between the dexamethasone-treated and untreated cochlear tissue using a microarray that covers 33 696 transcripts (24 878 genes) of mice and quantitative real-time RT-PCR. Results: The microarray analysis identified 39 genes that are up- or down-regulated by more than twofold in the presence of dexamethasone in the cochlear culture. Genes up- or down-regulated by at least threefold include Fkbp5, Gilz, glutathione peroxidase 3, claudin 10, glutamate-ammonia ligase, proteoglycan 1, integrin beta-like 1, and alpha subunit of glycoprotein hormone.

Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells

Objective To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method.Methods The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo.Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells.Co immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro.Results Positive blue clones were found in the QDO/X-α-gal plate.Eukaryotic expression vectors were co-transfected into HEK 293 cells,then HA-NLS RARα protein was immunoprecipitated by anti-HA polyclonal antibody,and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody.Conclusion The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.

Mutation screening of three candidate genes, ELOVL5, SMAP1 and GLULD1 in autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is the most common form of retinal dystrophy. It is featured by a great clinical and genetic heterogeneity. Different patterns of inheritance exist, such as autosomal dominant and recessive, X-linked and digenic. RP25, a locus for autosomal recessive retinitis pigmentosa (arRP), the most frequently inherited form of RP, was mapped to chromosome 6q between D6S257 and D6S1644 microsatellite markers. ELOVL5, SMAP1 and GLULD1 were selected on the basis of their location, tissue expression and/or function. ELOVL5 is implicated in the elongation of long chain fatty acids, including docosahexanoic acid (DHA), which constitutes 50% of the fatty acids of the outer segment of the photoreceptor. SMAP1 (stromal membrane associated protein 1) was found to be located within RP25 locus and is expressed in retina. GLULD1, glutamate-ammonia ligase (glutamine synthase) domain containing 1, plays a key role in the uptake and metabolism of glutamate in the retina. The absence of pathogenic mutations after molecular analysis argues against the implication of ELOVL5, SMAP1 and GLULD1 in the development of RP25 phenotype. Nevertheless, we could not rule them out as good candidates for other retinal degeneration mapping to the same chromosomal region.

Glutamine: commercially essential or conditionally essential? A critical appraisal of the human data

Glutamine is a nonessential amino acid that can be synthesized from glutamate and glutamic acid by glutamate-ammonia ligase. Glutamine is an important fuel source for the small intestine. It was proposed that glutamine is necessary for the maintenance of normal intestinal morphology and function in the absence of luminal nutrients. However, intestinal morphologic and functional changes related to enteral fasting and parenteral nutrition are less significant in humans than in animal models and may not be clinically significant. Therefore, it is unclear whether glutamine is necessary for the preservation of normal intestinal morphology and function in humans during parenteral nutrition. It was suggested that both glutamine-supplemented parenteral nutrition and enteral diets may pre-vent bacterial translocation via the preservation and augmentation of small bowel villus morphology, intestinal permeability, and intestinal immune function. However, it is unclear whether clinically relevant bacterial translocation even occurs in humans, much less whether there is any value in the prevention of such occurrences. Results of the therapeutic use of glutamine in humans at nonphysiologic doses indicate limited efficacy. Although glutamine is generally recognized to be safe on the basis of relatively small studies, side effects in patients receiving home parenteral nutrition and in those with liver-function abnormalities have been described. Therefore, on the basis of currently available clinical data, it is inappropriate to recommend glutamine for therapeutic use in any condition.

Pattern of akinete differentiation in the cyanobacteriumScytonema fritschii

The differentiation of akinetes inScytonema fritschii occurred adjacent to the newly developed heterocysts in late exponential phase. The filaments exhibited cell division leading to the formation of heterocysts, interspersed by the potential akinetes which could be identified by the accumulation of a large number of granules. Upon maturity, the akinetes acquired thick envelopes and were seen in elongated series interrupted by dead necridia which resulted from crumpling of the newly developed heterocysts. The formation of akinetes was accompanied by a change in color of cultures from blue-green to brown. Of the inorganic nitrogen sources tested, ammonium nitrate supported the formation of maximum percentage of akinetes. The incorporation of 7-azatryptophan and rifampicin in nitrate-free and nitrogen sources resulted in the production of heterocysts at a very high frequency in the late-exponential phase coinciding with akinete formation but the frequency of the latter was reduced. The activity of nitrogenase, nitrate reductase and glutamate-ammonia ligase was absent in mature akinetes. The absorption spectra of chlorophylla and phycobiliproteins revealed the presence of negligible amounts of the former white the latter were absent. The dry mass steadily increased during akinete differentiation with a concomitant decrease in C/N ratios.

Temperature-sensitive photosynthesis-deficient mutants of the cyanobacteriumAnabœna variabilis impaired in nitrogen assimilation

Temperature-sensitive (Ts) mutants ofAnabœna variabilis exhibited differences in the rates of oxygen evolution at 28 and 40°C. They were unable to perform photosynthesis and nitrogen fixation at 40°C beyond a period of 3 d in a nitrogen-deficient medium. However, the addition of combined nitrogen sources enhanced the growth of all the Ts mutants at both temperatures. Studies on nitrate uptake ty Ts mutants revealed the existence of a low affinity system, whereKm values were found to be lower than 1 mmol/L. The activity of nitrate reductase gradually increased with time at 28°C with the exception of Ts-161 where the activity decreased with time at 40°C. The rate of ammonia uptake byA. variabilis and its Ts mutants greatly differed and the results suggest the existence of a single phase of uptake. The activity of glutamate-ammonia ligase (GAL) of the parent and Ts mutants was slightly higher in cells from nitrogen-deficient medium when compared to nitrate grown cells at 28°C. At 40°C, the GAL activity decreased after 3 d. The inability of the Ts mutants to grow at 40°C appears to be due to an impairment in nitrogen asimilation.

Relationship of ammonia excretion, dinitrogen fixation and ammonia assimilatory enzymes to encystation in a continuous culture ofAzospirillum brasilense

Continuous culture studies have been carried out on a strain ofA. brasilense to study ammonia excretion, dinitrogen fixation and ammonia assimilatory enzymes (glutamate-ammonia ligase and glutamate synthase (NADPH)) in encysted conditions. High glutamate synthase (NADPH) and low ammonia excretion was observed at the time of induction of cyst formation. Low and oscillating nitrogenase activity was observed throughout the experiment.

Photoheterotrophic and chemoheterotrophic dinitrogen fixation and nitrate utilization by the cyanobacteriumAnabaena torulosa

Anabaena torulosa exhibited fructose-dependent growth, heterocyst differentiation and N2 fixation in nitrate-free (diazotrophic) cultures in photoheterotrophic and chemoheterotrophic conditions. The incorporation of nitrate into such cultures inhibited the formation of heterocysts and N2 fixation. The rate of NO 3 − uptake byA. torulosa in photoautotrophic, photoheterotrophic and chemoheterotrophic conditions was similar but it increased by 100% in phototrophic conditions. The activity of glucose-6-phosphate dehydrogenase was found to be maximum in phototrophic and photoheterotrophic conditions. Ferredoxin-NADP+ reductase, nitrate reductase and glutamate-ammonia ligase activities suggest that nitrate utilization takes place in nonphotosynthetic conditions.

53] Brain antioxidant activity of spin traps in Mongolian gerbils

Publisher Summary This chapter demonstrates that brain ischemia-reperfusion injury (IRI) results in significant increases in both salicylate hydroxylation and soluble brain protein oxidation, decreases in brain glutamine synthetase (glutamate-ammonia ligase) activity, and alterations in spontaneous exploratory activity. The IRI-mediated changes in behavior are accentuated in aged gerbils. Changes in IRI-mediated oxidation and behavioral dysfunction can be prevented by pretreatment with phenyl tert- butylnitrone (PBN) and related spin-trapping compounds. Based on the results obtained with PBN in IRI-mediated oxidation, it is determined if age-related alterations in brain function are be the result of progressive oxidative events. The chapter discusses that aged gerbils have a higher level of protein oxidation compared to young adult gerbils. As a result of these initial observations, the effects of daily PBN injections are evaluated on brain protein oxidation, glutamine synthetase activity, and radial arm maze performance. Radial arm maze performance is often used as an indicator of the status of short-term temporal-spatial memory in animals.

Detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer diseased patients: a potential diagnostic biochemical marker.

In this report, 8- and 2-azidoadenosine 5'-[gamma-32P]triphosphate were used to examine cerebrospinal fluid (CSF) samples for the presence of an ATP binding protein unique to individuals with Alzheimer disease (AD). A 42-kDa ATP binding protein was found in the CSF of AD patients that is not observed in CSF from normal patients or other neurological controls. The photolabeling is saturated with 30 microM 2-azidoadenosine 5'-[gamma-32P]triphosphate. Photoinsertion can be totally prevented by the addition of 25 microM ATP. Photoinsertion of 2-azidoadenosine 5'-triphosphate into the protein is only weakly protected by other nucleotides such as ADP and GTP, indicating that this is a specific ATP binding protein. A total of 83 CSF samples were examined in a blind manner. The 42-kDa protein was detected in 38 of 39 AD CSF samples and in only 1 of 44 control samples. This protein was identified as glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] based on similar nucleotide binding properties, comigration on two-dimensional gels, reaction with a polyclonal anti-GS antibody, and the presence of significant GS enzyme activity in AD CSF. In brain, GS plays a key role in elimination of free ammonia and also converts the neurotransmitter and excitotoxic amino acid glutamate to glutamine, which is not neurotoxic. The involvement of GS, if any, in the onset of AD is unknown. However, the presence of GS in the CSF of terminal AD patients suggests that this enzyme may be a useful diagnostic marker and that further study is warranted to determine any possible role for glutamate metabolism in the pathology of AD.

Colocalization in pericentral hepatocytes in adult mice and similarity in developmental expression pattern of ornithine aminotransferase and glutamine synthetase mRNA.

In situ hybridization showed that the mRNA for ornithine aminotransferase (OAT; ornithine-oxo-acid aminotransferase; L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13) colocalized with glutamine synthetase [GS; glutamate-ammonia ligase; L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] in pericentral hepatocytes of the adult mouse liver. In addition to an identical distribution in adult hepatocytes, OAT and GS have very similar expression patterns in fetal and neonatal liver. As was earlier described for GS, there is a low level of OAT mRNA in fetal cells and increasing pericentral levels in neonates that reach adult patterns within 2 weeks. These results suggest that the transcriptional regulation of the two genes is similar in the liver. However, there was a lack of colocalization of the mRNAs for the two enzymes in cells of the kidney, intestine, and brain, suggesting different regulatory decisions for the OAT and GS genes in the cells of these different tissues. The metabolic consequences of these localized expression patterns favor ammonia clearance from the blood by the liver and urea synthesis by the kidney.

Protein damage and lipid peroxidation: Effects of diethyl maleate, bromotrichloromethane and vitamin E on ammonia, urea and enzymes involved in ammonia metabolism

Changes in ammonia and urea were investigated as potential marker products of free radical damage to protein and subsequent metabolism of those damaged proteins in vivo. Both serum and liver lipid peroxidation products as measured by thiobarbituric-acid-reactive substances (TBARS) were increased by feeding rats a vitamin-E-deficient diet. The acute injection of diethyl maleate and bromotrichloromethane (DEM/ BrCCl 3) increased TBARS in liver of rats fed a vitamin-E-deficient diet. The concentrations of ammonia and urea in the serum and liver did not correlate with lipid peroxidation. The activities of liver glutaminase and arginase were decreased by DEM/BrCCl 3 treatment in rats fed vitamin-E-deficient diet. Glutamateammonia ligase activity was decreased by vitamin-E-deficient diet but not by DEM/BrCCl 3 treatment. Omithine carbamoyltransferase, arginosuccinate synthase, argininosuccinate lyase and glutamate dehydrogenase (NAD(P) +) were not affected by dietary vitamin E or by DEM/BrCCl 3. The data suggest that the concentrations of ammonia and urea, major by-products of nitrogen metabolism, are unchanged by the oxidant damage and lipid peroxidation, and that their control in vivo is a dynamic equilibrium of various metabolic pathways.

Assimilation of ammonia inParacoccus denitrificans

Two pathways serve for assimilation of ammonia in Paracoccus denitrificans. Glutamate dehydrogenase (NADP+) catalyzes the assimilation at a high NH4+ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH4+ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP+) and glutamate-ammonia ligase in P. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP+). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP+) by the high concentration of glutamine or its metabolic products as in the case when NH4+ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP+) was observed.

Factor influencing photoproduction of ammonia from dinitrogen byNostoc muscorum

Effective photoproduction of ammonia from dinitrogen has been achieved in a system consisting of a cell suspension of cyanobacteriumNostoc treated with a low concentration ofl-methionine sulfoximine (MSX). As a result of inactivation of cellular glutamate-ammonia ligase by MSX, growth was prevented, the rate of nitrogenase activity increased and about 90% of ammonia resulting from dinitrogen fixation was exported and accumulated in the ambient medium. The rate of NH4+ production was found to be regulated by different factors such as light-dark cycle, cell density, depth of culture and air bubbling. Ammonia production was stimulated by (i) a culture density corresponding to 1.5 μg chlorophyll a per mL, (ii) a depth of 10 mm, and (iii) continuous illumination for 24 h. The nitrogenase activity was found to be enhanced in the experimental sets where ammonia production was maximal.

Effect of tannin-rich leaves of oak (Quercus incana) on various microbial enzyme activities of the bovine rumen

1. The objective of the present experiment was to study the effects of oak (Quercus incana) leaves rich in tannins on various enzyme activities of the bovine rumen. 2. The procedure employed was incubation of tannin-rich, very-low-tannin or virtually tannin-free leaves in nylon-gauze bags in the rumen, and determination of enzyme activities in microbes tightly bound to the solid matrix and in microbes loosely plus tightly attached to the solid matrix. 3. The activities of urease (EC 3.5.1.5), carboxymethylcellulose, glutamate dehydrogenase (EC 1.4.1.2) and alanine aminotransferase (glutamic-pyruvic transaminase) (EC 2.6.1.2) were significantly lower in the tannin-rich group, whereas the activities of glutamate ammonia ligase (glutamine synthetase) (EC 6.3.1.2; both gamma-glutamyltransferase (EC 2.3.2.2) and the forward reaction) were higher in the tannin-rich group. These changes were more marked in micro-organisms tightly bound to the solid matrix than in the more complex microbial compartment. 4. The protein, DNA and RNA contents, and protein: RNA ratio, were significantly lower in the tannin-rich group, whereas no difference was observed for protein: DNA between the groups. 5. Effects of tannin-containing extracts of oak leaves on various rumen enzymes in vitro showed a trend similar to that observed in nylon-gauze bags, suggesting that the changes observed in various compartments were due to the tannins of oak leaves.