Glutamine Synthetase Protein Research Articles

GLT-1 transporter: An effective pharmacological target for various neurological disorders

l-Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) and is directly and indirectly involved in a variety of brain functions. Glutamate is released in the synaptic cleft at a particular concentration that further activates the various glutaminergic receptors. This concentration of glutamate in the synapse is maintained by either glutamine synthetase or excitatory amino acid proteins which reuptake the excessive glutamate from the synapse and named as excitatory amino acid transporters (EAATs). Out of all the subtypes GLT-1 (glutamate transporter 1) is abundantly distributed in the CNS. Down-regulation of GLT-1 is reported in various neurological diseases such as, epilepsy, stroke, Alzheimer's disease and movement disorders. Therefore, positive modulators of GLT-1 which up-regulate the GLT-1 expression can serve as a potential target for the treatment of neurological disorders. GLT-1 translational activators such as ceftriaxone are found to have significant protective effects in ALS and epilepsy animal models, suggesting that this translational activation approach works well in rodents and that these compounds are worth further pursuit for various neurological disorders. This drug is currently in human clinical trials for ALS. In addition, a thorough understanding of the mechanisms underlying translational regulation of GLT-1, such as identifying the molecular targets of the compounds, signaling pathways involved in the regulation, and translational activation processes, is very important for this novel drug-development effort. This review mainly emphasizes the role of glutamate and its transporter, GLT-1 subtype in excitotoxicity. Further, recent reports on GLT-1 transporters for the treatment of various neurological diseases, including a summary of the presumed physiologic mechanisms behind the pharmacology of these disorders are also explained.

Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.

Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.

Glutamine Synthetase Stability and Subcellular Distribution in Astrocytes Are Regulated by γ-Aminobutyric Type B Receptors

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

Glutamine synthetase plays a role in d-galactose-induced astrocyte aging in vitro and in vivo

Astrocytes play multiple roles in physiological and pathological conditions in brain. However, little is known about the alterations of astrocytes in age-related changes, and few aging models of the astrocytes in vitro have been established. Therefore, in the present study, we used d-galactose (D-Gal) to establish astrocyte aging model to explore the alterations of astrocytes in brain aging. We also used 1H nuclear magnetic resonance (1H NMR) spectra to verify the metabolic changes in the cerebral cortex of mice injected with D-gal. The results showed that D-gal (55mM) treatment for 1week induced senescence characteristics in cultured cortical astrocytes. Real-time PCR and western blot analysis showed that the levels of glutamine synthetase (GS) mRNA and protein were strikingly decreased in the cultured senescent astrocytes, and the senescent astrocytes showed less resistance to the glutamate-induced gliotoxicity. The impairments of glutamate–glutamine cycle and astrocytes were also found in the cerebral cortex of mice treatment with D-gal (100mg/kg) for 6weeks, and the level of GS mRNA was also found to be reduced markedly, being consistent with the result obtained from the senescent astrocytes in vitro. These results indicate that astrocyte may be the predominant contributor to the pathogenic mechanisms of D-gal-induced brain aging in mice, and GS might be one of the potential therapeutic targets of the aged brain induced by D-gal.

FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3'-UTR: possible contribution to ammonia detoxification.

Skeletal muscle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead-type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent reporter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3' region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skeletal muscle. In agreement, an intravenous ammonia challenge increased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle.

MP66-05 OXIDATIVE STRESS PROTEINS IDENTIFIED IN HUMAN SPERMATOZOA BY PROTEOMIC AND BIOINFORMATIC ANALYSIS

You have accessJournal of UrologyInfertility: Basic Research, Physiology & Pathophysiology1 Apr 2014MP66-05 OXIDATIVE STRESS PROTEINS IDENTIFIED IN HUMAN SPERMATOZOA BY PROTEOMIC AND BIOINFORMATIC ANALYSIS Rakesh Sharma, Ashok Agarwal, Damayanthi Durairajanayagam, Zhihong Cui, Ahmet Ayaz, and Edmund Sabanegh Rakesh SharmaRakesh Sharma More articles by this author , Ashok AgarwalAshok Agarwal More articles by this author , Damayanthi DurairajanayagamDamayanthi Durairajanayagam More articles by this author , Zhihong CuiZhihong Cui More articles by this author , Ahmet AyazAhmet Ayaz More articles by this author , and Edmund SabaneghEdmund Sabanegh More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2052AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Oxidative stress is associated with male infertility. Understanding the protein profile of spermatozoa is essential for better diagnosis of male infertility. The aim of the present study was to examine the effect of high ROS production on the proteomic profile and cellular distribution of spermatozoal proteins. METHODS Seminal ejaculates from 52 subjects (32 infertile men and 20 healthy men) were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis. Differentially (over- or under-) expressed proteins were identified and further categorized as common and unique. Functional bioinformatic analysis was done using both publicly available Gene Ontology annotations, as well as proprietary databases such as Ingenuity Pathway Analysis (IPATM) and MetacoreTM to identify the differentially affected processes, pathways, interactions and cellular distribution of the proteins. RESULTS Of 74 identified proteins, 47 were over-expressed and 27 under-expressed compared to the ROS- group. Fifteen of the 74 proteins were 2-fold differentially expressed in the ROS+ group, of which 10 were overexpressed and 5 underexpressed. Most of the differentially expressed proteins were localized to the cytoplasmic and intracellular regions. In the ROS+ group, there was increased expression of histone cluster 1H2ba, mitochondrial malate dehydrogenase precursor, transglutaminase 4, glutathione peroxidase 4 isoform A precursor, glutamine synthetase and heat shock proteins. Androgen receptor was identified as one of the topmost regulators in the ROS+ group with 21 differentially expressed proteins interacting with the receptor. cAMP responsive element modulator (CREM) signaling, which plays an important role in male fertility, was one of the key pathways associated with the differentially expressed proteins. CONCLUSIONS We identified various proteins that are implicated in a multitude of functions associated with response and management of oxidative stress. The increased expression of proteins in the ROS+ group suggests some of these proteins may serve as potential biomarkers of oxidative stress. Alterations reported in our study may help explain pathways leading to the altered semen phenotype, especially in men exhibiting oxidative stress. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e742 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Rakesh Sharma More articles by this author Ashok Agarwal More articles by this author Damayanthi Durairajanayagam More articles by this author Zhihong Cui More articles by this author Ahmet Ayaz More articles by this author Edmund Sabanegh More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

BackgroundSaccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins.ResultsTwo-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis.ConclusionsThis study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance the production yield of spinosad in future studies.

Diagnostic utility and limitations of glutamine synthetase and serum amyloid-associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma

Inflammatory hepatocellular adenoma can show overlapping histological features with focal nodular hyperplasia, including inflammation, fibrous stroma, and ductular reaction. Expression of serum amyloid-associated protein in inflammatory hepatocellular adenoma and map-like pattern of glutamine synthetase in focal nodular hyperplasia can be helpful in this distinction, but the pitfalls and limitations of these markers have not been established. Morphology and immunohistochemistry were analyzed in 54 inflammatory hepatocellular adenomas, 40 focal nodular hyperplasia, and 3 indeterminate lesions. Morphological analysis demonstrated that nodularity, fibrous stroma, dystrophic blood vessels, and ductular reaction were more common in focal nodular hyperplasia, while telangiectasia, hemorrhage, and steatosis were more common in inflammatory hepatocellular adenoma, but there was frequent overlap of morphological features. The majority of inflammatory hepatocellular adenomas demonstrated perivascular and/or patchy glutamine synthetase staining (73.6%), while the remaining cases had diffuse (7.5%), negative (3.8%), or patchy pattern of staining (15%) that showed subtle differences from the classic map-like staining pattern and was designated as pseudo map-like staining. Positive staining for serum amyloid-associated protein was seen in the majority of inflammatory hepatocellular adenomas (92.6%) and in the minority of focal nodular hyperplasia (17.5%). The glutamine synthetase staining pattern was map-like in 90% of focal nodular hyperplasia cases, with the remaining 10% of cases showing pseudo map-like staining. Three cases were labeled as indeterminate and showed focal nodular hyperplasia-like morphology but lacked map-like glutamine synthetase staining pattern; these cases demonstrated a patchy pseudo map-like glutamine synthetase pattern along with the expression of serum amyloid-associated protein. Our results highlight the diagnostic errors that can be caused by variant patterns of staining with glutamine synthetase and serum amyloid-associated protein in inflammatory hepatocellular adenoma and focal nodular hyperplasia.

Histological Mimicker of Hepatocellular Carcinoma in the Liver

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and the second most common fatal cancer in China and Southeast Asia. Introduction of new treatment modalities including targeted therapy may hopefully bring better survival of patients. A precise pathological diagnosis of HCC is crucial in view of the prognostic and treatment implications. The characteristic histology of HCC can be identified in most cases, yet histological mimickers and poorly differentiated tumors impose diagnostic challenges from time to time. The appropriate use of immunohistochemical markers and cautious interpretation of results are essential in difficult scenarios. Markers such as CD10, polyclonal carcinoembryonic antigen, HepPar-1, and the recently reported arginase 1 are helpful in confirming the hepatocytic differentiation of a malignant liver tumor. Glypican 3, either singly or in combination with glutamine synthetase and heat shock protein 70, is useful in distinguishing HCC from precursor lesions. Immunoexpression of CK19 may signify hepatic progenitor cell phenotype of HCCs. Application of specific immunohistochemical markers for other cell lineages would also help to confirm a diagnosis of liver metastasis. Clinicopathological correlation remains indispensible in pathological diagnosis.

A well-differentiated hepatocellular nodular lesion in the liver of a woman

The differential diagnoses of fine needle aspiration (FNA) biopsy of a well-differentiated hepatocellular nodule (WDHN) in a woman with hepatitis B cirrhosis are discussed. Radiological diagnosis was a large regenerative nodule containing fat. <h3>Points of interest</h3> Establish representative sampling. In cirrhosis, differential diagnosis include large regenerative nodule, dysplastic nodule (low- or high-grade), and early to progressed hepatocellular carcinoma (HCC). A nodule-in-nodule lesion occurs when there is malignant transformation in a benign parent nodule. Focal nodular hyperplasia and hepatocellular adenoma are less likely. Fatty change is common in WDHNs; diagnostic pitfalls include steatosis and focal fatty change. In WDHNs, the diagnostic dilemma is proof of malignancy. Serum α-fetoprotein has low sensitivity. Key cytological features in highly well-differentiated HCC: Generally cellular aspirates; monomorphous hepatocyte population with tendency to dispersion; small monotonous tumor cells with increased nuclear-cytoplasmic ratio, nuclear crowding, and scant thick cytoplasm; transgressing but no peripheral endothelium; and lack of bile duct cells. Polymorphous hepatobiliary population +/- restoration features (such ductules) favor benign nonneoplastic WDHN, after excluding non-representative sampling. Novel immunohistochemical markers such as glypican-3, glutamine synthetase and heat shock protein 70 may be helpful as an indicator of hepatocellular malignancy if any two markers are positive. Recommend combined cytohistological approach.

Effect of Subsoiling in Fallow Period and Nitrogen Application on Soil Moisture and Grain Protein Accumulation in Dryland Wheat

The objective of this study was to explore a technique to increase and maintain soil moisture of dryland through subsoiling during fallow period(SS) and the proper nitrogen(N) application in wheat production. In a two-year field experiment carried out in Wenxi County of Shanxi Province, China from autumn 2009 to summer 2011, we tested soil moistures in different soil layers, protein content and its components in grain, and activities of enzymes related to protein accumulation during grain filling after SS and non-tillage practice(CK) in combination with the applications of low(LN, 75 kg ha–1), medium(MN, 150 kg ha–1), or high(HN, 225 kg ha–1) N. Before sowing, the soil moistures in 0–300 cm soil layers under SS condition were higher than those under CK condition, especially in 60–160 cm soil layers, which were increased by 12%–34% in dry year and 10%–22% in humid year. Under SS condition, the activities of protein-related enzymes increased, such as glutamine synthetase(GS) in grain, glutamate synthase(GOGAT) in flag leaf, and GOGAT in grain in LN and MN treatments, and glutamic acid-pyruvate transaminase(GPT) in grain. In mature grains, the globulin content and protein yield in SS treatment were increased compared with CK, and Glutenin content and glutenin/gliadin ratio in humid year also increased in SS. The soil moisture in 20–200 cm soil layer at anthesis stage showed a decrease trend with the increase of nitrogen application level; however, the GS activity in grain, GOGAT activity in flag leaf at middle to late filling stage, GPT activity in grain at late filling stage were enhanced, and the contents of protein and its components in grain were increased. The glutenin/gliadin ratio in grain was the highest in MN treatment. Nitrogen application had obvious effects on soil moisture in the deep soil layer at anthesis, also on protein yield, GS and GPT activities in grainunder SS condition. Under SS plus N application condition, soil moisture at anthesis was closely correlated with grain albumin, glutenin, and protein content in humid year and with glutenin/gliadin ratio in dry year. In conclusion, subsoiling during fallow period is an effective method to maintain soil moisture in dryland; N application at 225 kg ha–1 has a positive effect on increasing contents of grain protein and its components, whereas N level of 150 kg ha–1 is favorable for high protein yield and glutenin/gliadin ratio in grain in dry year.

Molecular and biochemical modifications of liver glutamine synthetase elicited by daytime restricted feeding.

The circadian clock system in the liver plays important roles in regulating metabolism and energy homeostasis. Restricted feeding schedules (RFS) become an entraining stimulus that promotes adaptations that form part of an alternative circadian clock known as the food entrained oscillator (FEO). The aim of this study was to evaluate the daily variations of glutamine synthetase (GS) in liver under a daytime RFS. Hepatic GS properties were analysed at 3-h intervals over a 24-h period in adult male Wistar rats maintained in a 12:12 h light–dark cycle. RFS group: food access for 2-h in light phase, during 3 weeks. AL group: feeding ad libitum. Fa group: acute fast (21 h). Fa–Re group: acute fast followed by refed 2 h.mRNA expression was measured by RT-qPCR, protein presence by Western-blot and immunohistochemistry, enzyme activity by a spectrophotometric assay, and glutamine by high pressure liquid chromatography. Restricted feeding schedule induced circadian rhythmicity inmRNA levels of GS and the loss of the rhythmic pattern in mitochondrial GS activity. GS activity in liver homogenates displayed a robust rhythmic pattern in AL that was not modified by RFS. The presence of GS and its zonal distribution did not show rhythmic pattern in both groups. However, acute Fa and Fa–Re diminished GS protein and activity in liver homogenates. Hepatic glutamine concentrations showed a 24-h rhythmic pattern in both groups, in an antiphasic pattern. In conclusion, daytime RFS influences the liver GS system at different levels, that could be part of rheostatic adaptations associated to the FEO, and highlight the plasticity of this system.

Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana

The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%–48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%–43%), total GS activity (39%–45%) and soluble protein concentration (23%–24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.

Characterization of a glutamine synthetase gene DvGS1 from Dunaliella viridis and investigation of the impact on expression of DvGS1 in transgenic Arabidopsis thaliana

A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency.

A Sensing Role of the Glutamine Synthetase in the Nitrogen Regulation Network in Fusarium fujikuroi

In the plant pathogenic ascomycete Fusarium fujikuroi the synthesis of several economically important secondary metabolites (SM) depends on the nitrogen status of the cells. Of these SMs, gibberellin and bikaverin synthesis is subject to nitrogen catabolite repression (NCR) and is therefore only executed under nitrogen starvation conditions. How the signal of available nitrogen quantity and quality is sensed and transmitted to transcription factors is largely unknown. Earlier work revealed an essential regulatory role of the glutamine synthetase (GS) in the nitrogen regulation network and secondary metabolism as its deletion resulted in total loss of SM gene expression. Here we present extensive gene regulation studies of the wild type, the Δgln1 mutant and complementation strains of the gln1 deletion mutant expressing heterologous GS-encoding genes of prokaryotic and eukaryotic origin or 14 different F. fujikuroi gln1 copies with site-directed mutations. All strains were grown under different nitrogen conditions and characterized regarding growth, expression of NCR-responsive genes and biosynthesis of SM. We provide evidence for distinct roles of the GS in sensing and transducing the signals to NCR-responsive genes. Three site directed mutations partially restored secondary metabolism and GS-dependent gene expression, but not glutamine formation, demonstrating for the first time that the catalytic and regulatory roles of GS can be separated. The distinct mutant phenotypes show that the GS (1) participates in NH4 +-sensing and transducing the signal towards NCR-responsive transcription factors and their subsequent target genes; (2) affects carbon catabolism and (3) activates the expression of a distinct set of non-NCR GS-dependent genes. These novel insights into the regulatory role of the GS provide fascinating perspectives for elucidating regulatory roles of GS proteins of different organism in general.

AAV-Mediated, Optogenetic Ablation of Müller Glia Leads to Structural and Functional Changes in the Mouse Retina

Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.

Effects of recombinant human erythropoietin on the expression of glutamine synthetase of cultured retinal Miiller cells under high glucose condition

Objective To investigate the effect of recombinant human erythropoietin （rhEPO） on the expression of glutamine synthetase （GS） of cultured rat retinal Mtiller cells in high glucose environment in vitro. Methods Mtiller cells were isolated from retinas of 10 Sprague-Dawley rats at postnatal day three to five by trypsin digestion, and were randomly divided into six groups, including normal control group, high glucose group, high glucose ＋U/ml rhEPO group, high glucose＋10 U/ml rhEPO group, high glucose＋20 U/ml rhEPO group, high glucose＋ 40 U/ml rhEPO groups. After 48 hours, the apoptosis of retinal MUller cells were assayed by terminal transferase-mediated DNA end labelling assay, and the expression levels of GS protein were detected with semi-quantitative immunoeytochemistry. Results Compared with the normal control group, the cell viability and GS protein were reduced while the cell death increased in Mtiller cells cultured in high glucose, the difference was statistically significant （t= 27.4, P~0.01）. Compared with the high glucose group, rhEPO treatment reduced the apoptotic Mtiller cells （t= 857.2, 2 374.6, 2 473.2, 2 537.7; P〈0.01）, induced the expression of GS proteins （t=3.2, 18.0, 22.5, 26.4; P〈0.05）. Conclusions rhEPO can protect Muller cells from apoptosis under high glucose condition. The mechanism may be related to its function to up-regulate the GS protein expression, promote glutamic acid cycle, and reduce the excitotoxicity effects of high concentration of glutamate. Key words: Erythropoietin/pharmacology; Microglia; Glutamate-ammonia ligase

Novel aspects of glutamine synthetase (GS) regulation revealed by a detailed expression analysis of the entire GS gene family of Medicago truncatulaunder different physiological conditions

BackgroundGlutamine Synthetase (GS, EC 6.3.1.2) is a central enzyme in nitrogen metabolism, and a key component of nitrogen use efficiency (NUE) and plant yield and thus it is extremely important to understand how it is regulated in plants. Medicago truncatula provides an excellent model system to study GS, as it contain a very simple GS gene family comprising only four expressed genes, MtGS1a and MtGS1b encoding cytosolic polypeptides, and MtGS2a and MtGS2b encoding plastid-located enzymes. To identify new regulatory mechanisms controlling GS activity, we performed a detailed expression analysis of the entire GS gene family of M. truncatula in the major organs of the plant, over a time course of nodule or seed development and during a diurnal cycle.ResultsIndividual GS transcripts were quantified by qRT-PCR, and GS polypeptides and holoenzymes were evaluated by western blot and in-gel activity under native electrophoresis. These studies revealed that all four GS genes are differentially regulated in each organ of the plant, in a developmental manner, and identified new regulatory controls, which appear to be specific to certain metabolic contexts. Studies of the protein profiles showed that the GS polypeptides assemble into organ-specific protein complexes and suffer organ-specific post-translational modifications under defined physiological conditions. Our studies also reveal that GS expression and activity are modulated during a diurnal cycle. The biochemical properties of the four isoenzymes were determined and are discussed in relation to their function in the plant.ConclusionsThis work provides a comprehensive overview of GS expression and regulation in the model legume M. truncatula, contributing to a better understanding of the specific function of individual isoenzymes and to the identification of novel organ-specific post-translational mechanisms of GS regulation. We demonstrate that the GS proteins are modified and/or integrated into protein-complexes that assemble into a specific composition in particular organs of the plant. Taken together, the results presented here open new avenues to explore the regulatory mechanisms controlling GS activity in plants, a subject of major importance due to the crucial importance of the enzyme for plant growth and productivity.

Multifocal Hepatic Neoplasia in 3 Children With APC Gene Mutation

Hepatoblastoma (HB), the most common hepatic neoplasm in children is associated with germline mutations in adenomatous polyposis coli tumor-suppressor gene that cause familial adenomatous polyposis syndrome. Individuals with familial adenomatous polyposis have a 750 to 7500× the risk of developing HB. We report 3 children with APC gene mutation, who underwent resection or liver transplant for HB. In addition to HB, all 3 patients had multiple independent adenoma-like nodules lacking qualities of intrahepatic metastases. Twenty-five nodules were subjected to immunohistochemical analysis using a panel of antibodies including glypican-3 (GPC3), β-catenin, cytokeratin AE1/AE3, CD34, Ki-67, glutamine synthetase (GS), and fatty acid binding protein. The nodules were round, ranged in size from 0.2 to 1.5 cm, and paler than the background liver. All lacked the chemotherapy effect. The nodules were circumscribed but nonencapsulated and composed of well-differentiated hepatocytes with occasional minor atypical features and absent or rare portal tracts. One lesion displayed a "nodule-within-nodule" pattern. The nodules demonstrated diffuse GS overexpression. Nine (36%) nodules were focally reactive for GPC3, and 1 (4%) displayed focal nuclear β-catenin expression. The associated HB showed diffuse expression of GS, GPC3, and β-catenin nuclear staining. We interpret these nodules as neoplastic with most being adenomas (GPC3 negative) that show features of independent origin and represent early stages of carcinogenesis, implying potential to progress to HB or hepatocellular carcinoma. To our knowledge, this is the first report of multifocal neoplasms in patients with HB and APC gene mutation.

Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia

Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.