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Sanjay Kakar - One of the best experts on this subject based on the ideXlab platform.

  • genomic profiling of well differentiated hepatocellular neoplasms with diffuse Glutamine Synthetase staining reveals similar genetics across the adenoma to carcinoma spectrum
    Modern Pathology, 2019
    Co-Authors: Nancy M Joseph, Linda D Ferrell, Nafis Shafizadeh, Sarah E Umetsu, Sanjay Kakar
    Abstract:

    Well-differentiated hepatocellular neoplasms are currently classified in the World Health Organization scheme as hepatocellular adenoma or hepatocellular carcinoma. There is no recognized diagnostic category for atypical cases with borderline features, and we have designated these as atypical hepatocellular neoplasms. Diffuse Glutamine Synthetase staining is used as a surrogate marker to detect β-catenin activation, a well-recognized high risk feature in hepatocellular tumors. This study examined 27 well-differentiated hepatocellular neoplasms with diffuse Glutamine Synthetase staining, including 7 atypical hepatocellular neoplasms with no cytoarchitectural atypia, 6 atypical hepatocellular neoplasms with focal cytoarchitectural atypia, and 14 well-differentiated hepatocellular carcinomas. Capture-based next-generation sequencing was performed, and alterations in WNT pathway genes (CTNNB1, APC, AXIN1) were seen in 81% of cases (10/13 atypical hepatocellular neoplasms and 12/14 of hepatocellular carcinomas), while the molecular basis of diffuse Glutamine Synthetase staining was unclear in the remaining 19% of cases. Additional non-WNT pathway mutations (TP53, TSC1, DNMT3A, CREBBP) or copy number alterations were present in 56% of atypical hepatocellular neoplasms, with no significant difference in cases with or without focal cytoarchitectural atypia, supporting that all cases with β-catenin activation should be classified as atypical irrespective of atypia. Atypical hepatocellular neoplasm and hepatocellular carcinoma also demonstrated largely similar genomic profiles, but TERT promoter mutations were restricted to hepatocellular carcinoma (21%) and copy number alterations were more common in hepatocellular carcinoma (64 vs 31%). Mutational and copy number analysis may be helpful in characterization and risk stratification of atypical hepatocellular neoplasms when morphology and Glutamine Synthetase staining yield ambiguous results.

  • correlation of exon 3 β catenin mutations with Glutamine Synthetase staining patterns in hepatocellular adenoma and hepatocellular carcinoma
    Modern Pathology, 2016
    Co-Authors: Gillian Hale, Xinxin Liu, Li Che, David A Solomon, Christos G Tsokos, Nafis Shafizadeh, Xin Chen, Ryan M Gill, Sanjay Kakar
    Abstract:

    The current clinical practice is based on the assumption of strong correlation between diffuse Glutamine Synthetase expression and β-catenin activation in hepatocellular adenoma and hepatocellular carcinoma. This high correlation is based on limited data and may represent an oversimplification as Glutamine Synthetase staining patterns show wide variability in clinical practice. Standardized criteria for interpreting diverse Glutamine Synthetase patterns, and the association between each pattern and β-catenin mutations is not clearly established. This study examines the correlation between Glutamine Synthetase staining patterns and β-catenin mutations in 15 typical hepatocellular adenomas, 5 atypical hepatocellular neoplasms and 60 hepatocellular carcinomas. Glutamine Synthetase staining was classified into one of the three patterns: (a) diffuse homogeneous: moderate-to-strong cytoplasmic staining in >90% of lesional cells, without a map-like pattern, (b) diffuse heterogeneous: moderate-to-strong staining in 50-90% of lesional cells, without a map-like pattern, and (c) patchy: moderate-to-strong staining in 50% in both hepatocellular adenoma and hepatocellular carcinoma. The interpretation of β-catenin activation based on Glutamine Synthetase staining should be performed with caution, and the undetermined significance of various Glutamine Synthetase patterns should be highlighted in pathology reports.

  • diagnostic utility and limitations of Glutamine Synthetase and serum amyloid associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma
    Modern Pathology, 2014
    Co-Authors: Nancy M Joseph, Matthew M. Yeh, Linda D Ferrell, Michael Torbenson, Dhanpat Jain, Sanjay Kakar
    Abstract:

    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.

L L Sarlieve - One of the best experts on this subject based on the ideXlab platform.

  • Glutamine Synthetase expression and activity are regulated by 3 5 3 triodo l thyronine and hydrocortisone in rat oligodendrocyte cultures
    International Journal of Developmental Neuroscience, 1998
    Co-Authors: Dominique Baas, C Fressinaud, Ljubisa Vitkovic, L L Sarlieve
    Abstract:

    Abstract Glutamine Synthetase plays a central role in the detoxification of brain ammonia. Previously, we demonstrated that in vitro Glutamine Synthetase is expressed by all macroglial cell types and is developmentally regulated in oligodendrocyte lineage. Furthermore, Glutamine Synthetase is increased in secondary cultures of oligodendrocytes following a 72 h treatment with 30 nM 3,5,3′-triodo- l -thyronine [Baas, D., Bourbeau, D., Sarlieve, L. L., Ittel, M. E., Dussault, J. H. and Puymirat, J., Oligodendrocyte maturation and progenitor cell proliferation are independently regulated by thyroid hormone. Glia , 1997, 19, 324–332]. Hydrocortisone also increases Glutamine Synthetase activity after 72 h [Fressinaud, C., Weinrauder, H., Delaunoy, J. P., Tholey, G., Labourdette, G. and Sarlieve, L. L., Glutamine Synthetase expression in rat oligodendrocytes in culture: regulation by hormones and growth factors. J. Cell. Physiol. , 1991, 149, 459–468]; however, it is still unknown whether these increases in Glutamine Synthetase expression in oligodendrocytes after 3,5,3′-triodo- l -thyronine and hydrocortisone application are dose- and time-dependent. To further investigate this issue, we measured Glutamine Synthetase levels by Northern analysis, immunostaining and determination of Glutamine Synthetase activity after 3,5,3′-triodo- l -thyronine or hydrocortisone stimulation. We find that in rat oligodendrocyte secondary cultures, 3,5,3′-triodo- l -thyronine and hydrocortisone cause a dose- and time-dependent increase in Glutamine Synthetase mRNA, protein and activity. However, these hormones do not exert an additive or synergistic effect. Because purines, pyrimidines, and certain amino acids necessary for the synthesis of myelin components, are, in part, provided by the Glutamine Synthetase pathway, 3,5,3′-triodo- l -thyronine effect on myelination development and maturation could be mediated in part, through the Glutamine Synthetase gene regulation.

  • oligodendrocyte type 2 astrocyte o 2a progenitor cells express Glutamine Synthetase developmental and cell type specific regulation
    Molecular Psychiatry, 1998
    Co-Authors: Dominique Baas, D Dalencon, Ljubiša Vitković, C Fressinaud, L L Sarlieve
    Abstract:

    Oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells express Glutamine Synthetase: developmental and cell type-specific regulation

Antonio J Marquez - One of the best experts on this subject based on the ideXlab platform.

  • site directed mutagenesis of cys 92 from the α polypeptide of phaseolus vulgaris Glutamine Synthetase reveals that this highly conserved residue is not essential for enzyme activity but it is involved in thermal stability
    Plant Science, 2000
    Co-Authors: M T Clemente, Antonio J Marquez
    Abstract:

    The residue Cys-92 from the α-polypeptide of Phaseolus vulgaris Glutamine Synthetase is a highly conserved residue in prokaryotic and eukaryotic Glutamine Synthetase genes. This cysteine residue was previously proposed as a good candidate for being essential for enzyme activity. We have examined through heterologous expression in Escherichia coli and site-directed mutagenesis the functional importance of this residue. We have found that the thiol group of Cys-92 is not essential either for Glutamine Synthetase biosynthetic or transferase enzyme activities. The characteristic inhibition by p-hydroxymercuribenzoate (a specific sulphydryl reagent) was not substantially altered as a consequence of replacement of Cys-92 by Ala. Immunoreactivity of the Glutamine Synthetase mutant protein, examined both under native and denaturing conditions, was similar to the wild-type, indicating that no significant conformational changes were produced as a consequence of the introduced mutation. However, the mutant enzyme C92A was considerably less stable than the wild-type. These results indicate that Cys-92 is not an essential residue for enzyme activity but it is important for stability of the Glutamine Synthetase protein.

  • site directed mutagenesis of glu 297 from the alpha polypeptide of phaseolus vulgaris Glutamine Synthetase alters kinetic and structural properties and confers resistance to l methionine sulfoximine
    Plant Molecular Biology, 1999
    Co-Authors: M T Clemente, Antonio J Marquez
    Abstract:

    In this paper we examine the functionality of Glu-297 from the alpha-polypeptide of Phaseolus vulgaris Glutamine Synthetase (EC 6.3.1.2). For this purpose, the gln alpha cDNA was recombinantly expressed in Escherichia coli, and site-directed mutants constructed, in which this residue was replaced by alanine. The level of Glutamine Synthetase transferase catalytic activity in the mutant strain was 70-fold lower while biosynthetic activity remained practically unaffected. Kinetic parameters for both enzyme activities were not greatly altered except for the Km for ammonium in biosynthetic activity, which increased 100-fold. A similar result was reported when mutagenizing Glu-327 from E. coli Glutamine Synthetase, a residue shown to be present at the active site. This suggests that the Glu residue mutated in the higher-plant enzyme could develop a similar catalytic role to that of bacteria. Another characteristic feature of the mutant protein was its higher resistance to inhibition of the biosynthetic activity by L-methionine sulfoximine, a typical inhibitor of Glutamine Synthetase. In addition, we show that immunoreactivity of the Glutamine Synthetase mutant protein, both under native and denaturing conditions, is similar to the wild type, indicating that no deep conformational changes were produced as a consequence of the introduced mutation. However, structural changes in the active site can be predicted from alterations detected in the behaviour of the mutant protein towards affinity chromatography on 2',5'-ADP-Sepharose, as compared to the wild type. Nevertheless, complementation of an E. coli glnA mutation indicated that the E297A mutant enzyme was physiologically functional.

Lily Vardimon - One of the best experts on this subject based on the ideXlab platform.

  • basic fibroblast growth factor a potential inhibitor of Glutamine Synthetase expression in injured neural tissue
    Journal of Neurochemistry, 2001
    Co-Authors: Yelena Kruchkova, Iris Bendror, Avia Herschkovitz, Magda David, Avner Yayon, Lily Vardimon
    Abstract:

    Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of Glutamine Synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Muller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of Glutamine Synthetase expression. This inhibition appears to be mediated by the c-Jun protein which accumulated, in response to bFGF, exclusively in Muller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c-Jun protein and a decline in Glutamine Synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down-regulates Glutamine Synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down-regulation of Glutamine Synthetase.

  • a silencer element in the regulatory region of Glutamine Synthetase controls cell type specific repression of gene induction by glucocorticoids
    Journal of Biological Chemistry, 1999
    Co-Authors: Noa Avisar, Iris Bendror, Nadav Havazelet, Liora Shiftan, Lily Vardimon
    Abstract:

    Glutamine Synthetase is a key enzyme in the recycling of the neurotransmitter glutamate. Expression of this enzyme is regulated by glucocorticoids, which induce a high level of Glutamine Synthetase in neural but not in various non-neural tissues. This is despite the fact that non-neural cells express functional glucocorticoid receptor molecules capable of inducing other target genes. Sequencing and functional analysis of the upstream region of the Glutamine Synthetase gene identified, 5′ to the glucocorticoid response element (GRE), a 21-base pair Glutamine Synthetase silencer element (GSSE), which showed considerable homology with the neural restrictive silencer element NRSE. The GSSE was able to markedly repress the induction of gene transcription by glucocorticoids in non-neural cells and in embryonic neural retina. The repressive activity of the GSSE could be conferred on a heterologous GRE promoter and was orientation- and position-independent with respect to the transcriptional start site, but appeared to depend on a location proximal to the GRE. Gel-shift assays revealed that non-neural cells and cells of early embryonic retina contain a high level of GSSE binding activity and that this level declines progressively with age. Our results suggest that the GSSE might be involved in the restriction of Glutamine Synthetase induction by glucocorticoids to differentiated neural tissues.

  • Molecular control of Glutamine Synthetase expression in the developing retina tissue.
    Developmental Dynamics, 1993
    Co-Authors: Lily Vardimon, Iris Ben-dror, Nadav Havazelet, Lyle E. Fox
    Abstract:

    Glutamine Synthetase is a differentiation marker of the neural retina, whose expression is restricted to Muller glia cells, is inducible by glucocorticoids and is dependent on tissue development. The retina tissue acquires the competence to express GS in response to glucocorticoids with development, although the level of hormone binding activity in the cells does not alter with age. Using CAT constructs that are controlled by “simple GRE” promoters we demonstrated that glucocorticoid receptor transcription activity in retina cells increases with development. The increase in receptor activity correlates directly with the increase in inducibility of the Glutamine Synthetase gene and inversely with the rate of retina cell proliferation. At early developmental ages, when retina cells are still proliferating, the glucocorticoid receptor is transcriptionally inactive and Glutamine Synthetase expression cannot be induced. Receptor activity increases progressively with development and by day 12, when cell proliferation ceases, competence for Glutamine Synthetase induction is high. This competence for Glutamine Synthetase induction can be repressed by overexpressing the oncogene v-src, which stimulates retina cell proliferation. We discuss possible mechanisms for developmental-dependent modulation of glucocorticoid receptor transcriptional activity. © 1993 wiley-Liss, Inc.

Lourdes Humanes - One of the best experts on this subject based on the ideXlab platform.

  • purification and characterization of Glutamine Synthetase from the green alga monoraphidium braunii
    Plant Science, 1997
    Co-Authors: Jose Manuel Garciafernandez, Antonio Lopezruiz, Lourdes Humanes, Jesus Diez Dapena
    Abstract:

    Abstract Glutamine Synthetase (EC 6.3.1.2] has been purified from the green alga Monoraphidium braunii. The enzyme was purified by a method which included consecutive chromatographies on: DEAE Sepharose, Blue Sepharose, second DEAE Sepharose, Sephacryl S-300 and Phenyl Sepharose CL-4B. The apparent molecular weight of the GS subunit was approximately 42 000. Since the undissociated enzyme has a molecular weight of 295 000, M. braunii GS can be considered as a plant type GS, probably with octameric structure. Purified Glutamine Synthetase was inhibited by some amino acids and nucleotides. The Stokes radius of the native enzyme was 6.13 nm. Values for apparent Michaelis constants of the physiological activity of the purified enzyme for glutamate, ATP, and ammonium were 5.7, 0.85 and 0.05 mM respectively. Alanine, glycine, aspartate and serine inhibited both transferase and synthetic activities of GS. Glutamine Synthetase from M. braunii was inhibited by p-hydroxymercuribenzoate, the effect being reversed by treatment with dithioerytritol.

  • Glutamine Synthetase from the green alga monoraphidium braunii is regulated by oxidative modification
    Plant Science, 1995
    Co-Authors: Lourdes Humanes, Antonio Lopezruiz, Jose Manuel Garciafernandez, Jesús Diez
    Abstract:

    Monoraphidium braunii Glutamine Synthetase is inactivated by several mixed-function oxidation systems. Inactivation requires oxygen and a metal cation as it does not take place under anaerobic conditions or in the presence of EDTA. Glutamine Synthetase can be protected against that inactivation by peroxidase and catalase but not by superoxide dismutase indicating that hydrogen peroxide is involved in the process, although hydrogen peroxide is not itself effective. The oxidative modification of Glutamine Synthetase renders the protein more sensitive to temperature and susceptible to proteolytic attack. This has been demonstrated by measuring by quantitative immunoelectrophoresis the levels of Glutamine Synthetase antigen, in enzymatic preparations treated with different oxidation systems. Besides, immunoblotting of crude extracts in the presence of mixed-function oxidation systems shows the disappearance of material cross-reacting with anti-Glutamine Synthetase antibodies. Other results show that Glutamine Synthetase from Chlamydomonas reinhardtii could be subjected to the same kind of oxidative inactivation. The possible regulatory role of oxidative modification of Glutamine Synthetase in green algae is discussed.