Glutamine Synthetase

Field studies were conducted in Johnson Key Basin, Florida Bay, USA from September 2002 through September 2004 to examine physiological, ecological, and behavioral characteristics of the gulf toadfish, Opsanus beta (Goode and Bean in Proc US Natl Mes 3:333-345, 1880), in relation to nitrogen metabolism, habitat usage, and spawning. Fish collected 5 cm above sediments in experimental shelters (epibenthic) were compared with those collected by throw traps which were found on or burrowing within sediments. The relationship between microhabitat ammonia and urea excretion, as determined by the enzymatic activity of glutamine synthetase (GS), was examined. The hypothesis tested was that O. beta occupying epibenthic nests were less ureotelic with lower GS activities than non-nesting individuals on/in sediments, due to a decreased environmental ammonia burden. Porewater total ammonia (T Amm ) concentrations at a sediment depth of 5 cm, i.e., the approximate depth of burrowing toadfish, ranged from 0 to 106.5 lmol N l -1 while the pH ranged from 7.48 to 9.14. There was a weak but significant correlation between environmental ammonia (NH 3 ) concentration and hepatic GS activity for epibenthic toadfish (P < 0.001, r 2 = 0.10), but not for burrowing toadfish. Mean urea-N and T Amm concentrations within shelters occupied by toadfish (n = 281) were 9.8 ± 0.83 lmol N l -1 and 13.0 ± 0.7 lmol N l -1 , respectively. As predicted, hepatic GS activity was significantly lower in epibenthic toadfish captured in shelters (4.40 ± 0.24 lmol min -1 g -1 ; n = 281) as compared to individuals on/in sediments (6.61 ± 0.47 lmol min -1 g -1 ; n = 128). Glutamine synthetase activity generally peaked in March (spawning season) and was lowest in July. Gender differences in hepatic and branchial GS activity were also found during the spawning season, which is attributable to the fact that males brood and guard offspring in their epibenthic nests while females often rest on or burrow into the sediments. Finally, hepatic and branchial GS appeared to have different patterns of enzymatic activity suggesting functional differences in gene expression.

Hepatocellular nodules in cirrhosis include regenerative (large regenerative, LRN) and dysplastic (low and high grade, LGDN and HGDN) nodules, early and grade 1 HCC (eHCC-G1), and overt HCC. The differential diagnosis may be particularly difficult when lesions such as HGDN and eHCC-G1 are involved. We investigated the diagnostic yield of a panel of 3 putative markers of hepatocellular malignancy such as HSP70, glypican 3 (GPC3), and glutamine synthetase (GS). We selected 52 surgically removed nonmalignant nodules (15 LRNs, LGDNs, 22 HGDNs) and 53 HCCs (10 early, 22 grade 1, and 21 grade 2-3) and immunostained them for HSP70, GPC3, and GS. The sensitivity and specificity of the individual markers for the detection of eHCC-G1 were 59% and 86% for GS, 69% and 91% for GPC3, and 78% and 95% for HSP70. We identified 2 main phenotypes: (1) all negative, seen in 100% LRN and LGDN, 73% HGDN and 3% eHCC-G1; (2) all positive, a feature detected in less than half the eHCC-G1. Using a 3-marker panel, when at least 2 of them, regardless which, were positive, the sensitivity and specificity for the detection of eHCC-G1 were respectively 72% and 100%; the most sensitive combination was HSP70؉/GPC3؉ (59%) when a 2-marker panel was used. Conclusion: The adopted panel of 3 markers is very helpful in distinguishing eHCC-G1 from dysplastic nodules arising in cirrhosis. (HEPATOLOGY 2007;45:725-734.) Abbreviations: eHCC-G1, early and grade 1 HCC; GPC3, glypican 3; GS, glutamine synthetase; HGDN, high grade dysplastic nodule; HSP70, heat shock protein 70; LGDN, low grade dysplastic nodule; LRN, large regenerative nodule. From the departments of 1 Pathology and

Glutamine synthetase (GS) has long been known to be expressed exclusively in pericentral hepatocytes most proximal to the central veins of liver lobuli. This enzyme as well as its peculiar distribution complementary to the periportal compartment for ureogenesis plays an important role in nitrogen metabolism, particularly in homeostasis of blood levels of ammonium ions and glutamine. Despite this fact and intensive studies in vivo and in vitro, many aspects of the regulation of its activity on the protein and on the genetic level remained enigmatic. Recent experimental advances using transgenic mice and new analytic tools have revealed the fundamental role of morphogens such as wingless-type MMTV integration site family member signals (Wnt), beta-catenin, and adenomatous polyposis coli in the regulation of this particular enzyme. In addition, novel information concerning the structure of transcription factor binding sites within regulatory regions of the GS gene and their interactions with signalling pathways could be collected. In this review we focus on all aspects of the regulation of GS in the liver and demonstrate how the new findings have changed our view of the determinants of liver zonation. What appeared as a simple response of hepatocytes to blood-derived factors and local cellular interactions must now be perceived as a fundamental mechanism of adult tissue patterning by morphogens that were considered mainly as regulators of developmental processes. Though GS may be the most obvious indicator of morphogen action among many other targets, elucidation of the complex regulation of the expression of the GS gene could pave the road for a better understanding of the mechanisms involved in patterning of liver parenchyma. Based on current knowledge we propose a new concept of how morphogens, hormones and other factors may act in concert, in order to restrict gene expression to small subpopulations of one differentiated cell type, the hepatocyte, in different anatomical locations. Although many details of this regulatory network are still missing, and an era of exciting new discoveries is still about to come, it can already be envisioned that similar mechanisms may well be active in other organs contributing to the fine-tuning of organ-specific functions.

The bulk 15 N-value of plant (leaf) biomass is determined by that of the inorganic primary nitrogen sources NO 3 À , NH 4 + and N 2 , and by isotope discriminations on their uptake or assimilation. NH 4 + from these is transferred into ''organic N'' mainly by the glutamine synthetase reaction. The involved kinetic nitrogen isotope effect does not become manifest, because the turnover is quantitative. From the product glutamine any further conversion proceeds in a ''closed system'', where kinetic isotope effects become only efficient in connection with metabolic branching. The central and most important corresponding process is the GOGAT-reaction, involved in the de novo nitrogen binding and in recycling processes like the phenylpropanoid biosynthesis and photorespiration. The reaction yields relatively 15 N-depleted glutamate and remaining glutamine, source of 15 N-enriched amide-N in heteroaromatic compounds. Glutamate provides nitrogen for all amino acids and some other compounds with different 15 Nabundances. An isotope equilibration is not connected to transamination; the relative 15 N-value of individual amino acids is determined by their metabolic tasks. Relative to the bulk 15 N-value of the plant cell, proteins are generally 15 N-enriched, secondary products like chlorophyll, lipids, amino sugars and alkaloids are depleted in 15 N. Global 15 N-values and 15 N-patterns of compounds with several N-atoms can be calculated from those of their precursors and isotope discriminations in their biosyntheses.

Three fish species of the family Batrachoididae, the gulf toadfish (Opsanus beta), the oyster toadfish (Opsanus tau), and the plainfin midshipman (Porichthys notatus) demonstrated exceptionally high tolerances to elevated water ammonia with 96-h LC50 values of 9.75, 19.72 and 6 mM total ammonia, respectively. Using pH values we calculated the corresponding unionized ammonia (NH 3 ) values to be 519, 691 and 101 mM, respectively. These values are well above typical values for most teleost fishes, but close to those of ureotelic fish examined to date. Following sublethal high ammonia exposure (HAE) blood and tissue (brain, liver and muscle) sampling confirmed that internal ammonia levels rose substantially in all three species, suggesting that they were not simply avoiding toxicity by impermeance to ammonia. The three species of batrachoidids can be characterized in the following manner with respect to the inabilities to synthesize and excrete urea, based on these studies and prior research: O. beta (fully ureotelic)\O. tau (moderately ureotelic) \P. notatus (ammoniotelic). While some of the high ammonia tolerance for O. beta and O. tau can be explained by their ability to detoxify it to urea, other mechanisms must be at play for P. notatus. Further experiments determined that all three species possess rather high activities of glutamine synthetase (GSase) in brain especially (60-180 U g − 1 ), that glutamine accumulates in many tissues, and that LC50 values are correlated positively with brain GSase activity. Taken together, our results suggest that alternative/additional mechanisms for ammonia detoxification via urea synthesis must be considered to explain the exceptionally high ammonia tolerance of this group.

We were interested in the effects of immobilization (IM), a single bout of exercise (E) and immobilization followed by exercise (EIM) on memory and oxidative damage of macromolecules in hippocampus of rat brain. Eight hours of IM resulted in impairment of passive avoidance test (memory retrieval deficit) and increased latency to start locomotion in an open-field test. Two hours of

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nieotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C16) from a tobacco )~gtl 1 expression library. A longer Fd-GOGAT cDNA clone (C35) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco )~gtl0 cDNA library using C16 as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C35 was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.

© M a s s o n , P a r i s , 2 0 0 4 A n n P a t h o l 2 0 0 4 ; 2 4 : 5 3 6 -5 5 Fabre M, Yilmaz F, Annick Buendia M. Les tumeurs hépatiques de l'enfant : expérience à propos de 245 tumeurs et revue de la littérature. Ann Pathol 2004 ; 24 : 536-55.

© M a s s o n , P a r i s , 2 0 0 4 A n n P a t h o l 2 0 0 4 ; 2 4 : 5 3 6 -5 5 Fabre M, Yilmaz F, Annick Buendia M. Les tumeurs hépatiques de l'enfant : expérience à propos de 245 tumeurs et revue de la littérature. Ann Pathol 2004 ; 24 : 536-55.

The objective of the present work was to determine the effect of NH4NO3 toxicity on nitrogen metabolism in Phaseolus vulgaris L. cv. Strike. The NH4NO3 was applied to the nutrient solution at 6 (N1), 12 (N2), 18 (N3), and 24 mM (N4). The availability of NO3− and NH4+ in the roots and leaves of green bean proved to be influenced by the different NH4NO3 treatments, being the NO3− and NH4+ translocated towards the aerial part of the plant and subsequently assimilated in the leaves. The most important enzymatic activities within nitrogen metabolism, such as nitrate reductase, nitrite reductase, glutamine synthetase and glutamate synthase were affected negatively by the highest NH4NO3 rate (N4). Finally, in our experiment, possibly the high accumulation of NO3−, and principally of NH4+, in both organs due to the application of high NH4NO3 rates could be the direct causes of the reduced biomass production in plants and of the reduction of N assimilation process in roots and leaves, indicating that green bean plants are highly sensitive to high N levels.

While earthworms are known to improve plant growth by improving the structure of the soil, recent work has suggested that earthworms also produced humic substances endowed with hormone-like activity. Suspensions of Daucus carota (carrot) cells were treated with auxin derivatives (2,4-D=2,4-dichlorophenoxyacetic acid, IAA=indole-3-acetic acid and NAA=1naphthylacetic acid) and a humic substance of low molecular weight (HEf), obtained from the faeces of the earthworms Nicodrilus caliginosus and Allolobophora rosea, which have auxin-like activity. HEf, at a concentration of 200 mg C l Àl , caused an increase in carrot cell growth, determined by packed cell volumes, similar to that of 2,4-D, and morphological changes induced by HEf were similar to those induced by IAA. Humic matter and the tested auxin derivatives stimulated carrot cell glutamate dehydrogenase (GDH), glutamine synthetase (GS) and malic dehydrogenase (MDH) to various extents. When carrot cells were treated with HEf or IAA, GDH and MDH isoenzymes separated by isoelectrofocusing showed similar polymorphism. The addition of HEf or auxin derivatives to carrot cells caused an increase in free amino acids belonging to the oxaloacetate and aketoglutarate pathways. Our results showed that HEf aects nitrate metabolism, has auxin-like activity, was strongly acidic and had elevated peptide, carbohydrate and aliphatic C contents. Our work has con®rmed that the eective materials in the HEf were highly acidic and of low molecular weight. We conclude that, in addition to the already well established modes of action, earthworms are important in humogenesis because their faeces contained humic substances that can in¯uence plant growth via physiological eects. #

Metagenomics projects based on shotgun sequencing of populations of microorganisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium-and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

To better understand some basic aspects of the nitrogen economy in Posidonia oceanica and, specifically, the seasonality of the processes of storage, translocation and assimilation, we examined nitrogen speciation into soluble compounds, both inorganic (nitrates, nitrites and ammonium) and organic (free amino acids, FAA, and total soluble protein, TSP), and the nitrogen assimilation potential (through the glutamine synthetase activity measurement) in the leaves, rhizomes and roots of P. oceanica over a 1-year cycle. Only a limited amount of inorganic nitrogen was found, accounting for less than 3.3% of the total nitrogen content, and it was mostly in the form of ammonium. Nitrate and nitrite concentrations were very low, always below 7.2 Amol g À 1 dw in annual average. Among the organic soluble fractions, FAAs were the most abundant, accounting for up to 50% of N pools. Rhizomes were the organs in which FAA concentrations reached their maximum value. The leaves showed higher nitrogen assimilation potential than the roots and this assimilation potential was highest during and after the period of maximum leaf growth, probably corresponding to the assimilation of both new and recycled nitrogen. Our results suggest that 5% of the total nitrogen assimilation occurs in roots and 79% in leaves on an annual average. In addition, rhizomes contributed to the total shoot nitrogen assimilation by 32 -54% between autumn and spring. Rhizomes appear as key organs in the nitrogen economy of the plant, not only as a major site for nitrogen assimilation but also as an organ for nitrogen storage. This storage, mostly in the form of FAA, occurs during periods of high availability and low demand (winter). This stored nitrogen can supply up to 33% of plant demands during the moment of maximum leaf growth (i.e. late spring). D

Mercury(II) or Hg2+, is neurotoxic. When exposed to normal brain tissue homogenates, neurons in culture, Hg2+ (mercury(II) or mercuric mer- cury) is capable of causing many of the same biochemical aberrancies found in Alzheimer&#39;s diseased (AD) brain. Also, rats exposed to mercury(0), metallic mercury, vapor show some of these same abnormalities in their brain tissue. Specifically, the rapid inactivation of

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

In the present study, two fresh water fishes namely, Channa punctatus and Clarias batrachus, were exposed to three sub-acute concentrations of synthetic pyrethroid, cypermethrin, for 96 h to evaluate the role of amino acids in fulfilling the immediate energy needs of fishes under pyrethroid induced stress as well as to find out the mechanism of ammonia detoxification. The experiments were designed to estimate the levels of free amino acid, urea, ammonia and the activities of aspartate aminotransferase (AAT), alanine aminotransferase (AlAT), glutamate dehydrogenase (GDH), glutamine synthetase (GS) and arginase in some of the vital organs like brain, gills, liver, kidney and muscle of both fish species. The significant decrease in the levels of amino acids concomitant with remarkable increase in the activities of AAT, AlAT and GDH in these vital tissues of fish species elucidated the amino acid catabolism as one of the main mechanism of meeting out the immediate energy demand of the fishes in condition of cypermethrin exposure. The levels of ammonia were significantly increased at 10% of 96 h LC 50 of cypermethrin in the different organs such as brain, gills, liver, kidney and muscle of both fish species while 15% and 20% concentrations of 96 h LC 50 of cypermehrin registered remarkable decline in both fish species. The differential increment in the activities of GDH, GS and arginase and in the level of urea established three different alternative mechanisms of ammonia detoxification. The results indicated that in C. punctatus, the prevalent mode of nitrogen excretion is in the form of conversion of ammonia into glutamine and glutamate while in C. batrachus, the excessive nitrogen is excreted in the form of urea synthesized from ammonia.

We present evidence, for the first time, of the occurrence of a transport system common for amino acid methionine, and methionine/glutamate analogues L-methionine-DL-sulfoximine (MSX) and phosphinothricin (PPT) in cyanobacterium Nostoc muscorum. Methionine, which is toxic to cyanobacterium, enhanced its nitrogenase activity at lower concentrations. The cyanobacterium showed a biphasic pattern of methionine uptake activity that was competitively inhibited by the amino acids alanine, isoleucine, leucine, phenylalanine, proline, valine, glutamine, and asparagine. The methionine/glutamate analogue-resistant N. muscorum strains (MSX-R and PPT-R strains) also showed methionine-resistant phenotype accompanied by a drastic decrease in 35 S methionine uptake activity. Treatment of protein extracts from these mutant strains with MSX and PPT reduced biosynthetic glutamine synthetase (GS) activity only in vitro and not in vivo. This finding implicated that MSX-and PPT-R phenotypes may have arisen due to a defect in their MSX and PPT transport activity. The simultaneous decrease in methionine uptake activity and in vitro sensitivity toward MSX and PPT of GS protein in MSX-and PPT-R strains indicated that methionine, MSX, and PPT have a common transport system that is shared by other amino acids as well in N. muscorum. Such information can become useful for isolation of methionine-producing cyanobacterial strains.

We analyzed the effect of omission of sulfur (S) from the nutrient solution and then restoration of S-source on the uptake and assimilation of nitrate in rapeseed. Incubation in nutrient solution without S for 1-6 days led to decline in uptake of nitrate, activities, and expression levels of nitrate reductase (NR) and glutamine synthetase (GS). The nitrite reductase (NiR) and glutamate synthase (GOGAT) activities were not considerably affected. There was significant enhancement in nitrate content and decline in sulfate content. Evaluation of amino acid profile under S-starvation conditions showed two-to fourfold enhancement in the contents of arginine, asparagine and O-acetyl-L-serine (OAS), whereas the contents of cysteine and methionine were reduced heavily. When the S-starved plants were subjected to restoration of S for 1, 3, 5, and 7 days, activities and expression levels of NR and GS recovered within the fifth and seventh days of restoration, respectively. Exogenous supply of metabolites (arginine, asparagine, cysteine, glutamine, OAS, and methionine) also affected the uptake and assimilation of nitrate, with a maximum for OAS. These results corroborate the tight interconnection of S-nutrition with nitrate assimilation and that OAS plays a major role in this regulation. The study must be helpful in developing a nutrient-management technology for optimization of crop productivity.

To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two dierent approaches were conducted using the model legume Lotus japonicus. In the ®rst series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to a decrease in plant biomass production. Using 15 N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using a series of recombinant inbred lines issued from the crossing of two dierent Lotus ecotypes, Gifu and Funakura. It was con®rmed that a negative relationship exists between root GS expression and plant biomass production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least 13% of plant growth variation can be accounted for by variation in GS activity.

Here, we study the effect caused by three trace elements-Al, Se, and Mo-applied at the same concentration (100 mM) and in their oxyanionic forms-NaAl(OH) 4 , Na 2 SeO 4 , and Na 2 MoO 4 -on NO 3 À assimilation (NO 3 À , nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) activities, and concentrations of amino acids and proteins) in sunflower (Helianthus annuus L. var. Kasol) plants. The most harmful element for sunflower plants proved to be selenate, followed by aluminate. On the contrary, the application of molybdate had no negative effect on the growth of this plant, suggesting the possibility of using sunflower for the phytoremediation of this metal, mainly in agricultural zones used for grazing where the excess of this element can provoke problems of molybdenosis in ruminants (particularly in cattle). In addition, we found that the alteration of NO 3 À assimilation by SeO 4 2À and Al(OH) 4 À directly influences the growth and development of plants, foliar inhibition of NR activity by SeO 4 2À being more harmful than the decrease in foliar availability of NO 3 À provoked by Al(OH) 4 À .

Glutamine synthetase (GS; E.C.6.3.1.2) is a key enzyme in higher plants with two isozymes; cytosolic GS1 and plastidic GS2, which involves in assimilation and recycling of NH 4 + ions and maintenance of complex traits such as crop nitrogen use efficiency (NUE) and yield. Our present understanding of crop-NUE and its correlation with the GS family gene's functional role is inadequate that hinders harnessing the benefit of this key enzyme in crop improvement. In this report, we performed a comprehensive investigation on the phylogenetic relationship, structural properties, complex multilevel gene regulation, and expression patterns of the GS genes to enrich present understanding about the enzyme. Our Gene Ontology (GO) and protein-protein interactions (PPI) analysis reveal functional aspects of GS isozymes, in stress mitigation, aging, nucleotide biosynthesis/transport, DNA repair, and response to metals. The insight gained herein put forth a step forward to the future research strategies in developing climate-smart crops for global sustainability. Box 1: Summary • Phylogenetic analysis of 447 Glu-Synt domain-containing protein sequences from 64 higher plants species clustered into two major clades; Clade I and Clade II.  Plants genome comprises a diverse repository sequence of Glu-Synt domain-containing proteins, among which 53% are of cytosolic GS1 and plastidic GS2, remaining are members of GS-like (9%), FluG-related (9%), computationally recognized GS (4%), NodGS (3%) family and uncharacterized (22%).  Structural modeling of GS isozymes showed that amino acid residues associated with same function are mostly conserved in plant system.  Multi-level gene regulations of GS genes are strongly influenced by the cellular and environment factors. • Present data-mining study revealed that GS isozymes are associates with different important traits including abiotic stress mitigation, aging, promoting nucleotide biosynthesis/transport, and DNA-repair in the plant system.

Cyanobacterial populations introduced into crop fields as biofertilizer become non-target organisms for the pesticides and fungicides applied in the field. Effect of four commonly used pesticides viz. Bagalol, Mancozeb (fungicides), Thiodan and Phorate (insecticides) was studied on growth and different enzymes of four cyanobacterial species viz. Nostoc ellipsosporum, Scytonema simplex, Tolypothrix tenuis, and Westiellopsis prolifica. EC 50 concentration of each pesticide was determined for all cyanobacteria. Bagalol and Thiodan were found to be the most toxic. Both the fungicides and insecticides inhibited the activity of nitrogenase and glutamine synthetase (GS) at EC 50 concentration in all the four species studied. Bagalol incurred maximum inhibition of nitrogenase and GS activity on N. ellipsosporum and S. simplex while Thiodan and Phorate had maximum effect on T. tenuis, and W. prolifica. Mancozeb had lesser effect on all the above enzymes. One catabolic enzyme of carbohydrate metabolism, isocitrate dehydrogenase (ICDH) and one anabolic enzyme isocitrate lyase (ICL), which is related to glyoxylate pathway as well as gluconeogenesis, were also assayed. Cell free extracts of cyanobacteria treated with pesticides for 7 days show a drastic reduction of ICDH activity. ICL activity was induced in the organisms when treated with pesticides.

In the present study, N and S assimilation, antioxidant enzymes activity, and yield were studied in N and S-treated plants of Brassica juncea (L.) Czern. & Coss. (cvs. Chuutki and Radha) under salt stress. The treatments were given as follows: (1) NaCl 90 mM+N 0 S 0 mg kg -1 sand (control), (2) NaCl 90 mM+N 60 S 0 mg kg -1 sand, (3) NaCl 90 mM+N 60 S 20 mg kg -1 sand, (4) NaCl 90 mM+N 60 S 40 mg kg -1 sand, and (5) NaCl 90 mM+N 60 S 60 mg kg -1 sand. The combined application of N (60 mg kg −1 sand) and S (40 mg kg −1 sand) proved beneficial in alleviating the adverse effect of salt stress on growth attributes (shoot length plant −1 , fresh weight plant −1 , dry weight plant −1 , and area leaf −1 ), physio-biochemical parameters (carbonic anhydrase activity, total chlorophyll, adenosine triphosphate-sulphurylase activity, leaf N, K and Na content, K/Na ratio, activity of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, catalase, superoxide dismutase, ascorbate peroxidase and glutathione reductase, and content of glutathione and ascorbate), and yield attributes (pods plant −1 , seeds pod −1 , and seed yield plant −1 ). Therefore, it is concluded that combined application of N and S induced the physiological and biochemical mechanisms of Brassica. The stimulation of antioxidant enzymes activity and its synergy with N and S assimilation may be one of the important mechanisms that help the plants to tolerate the salinity stress and resulted in an improved yield.

The main findings of a cooperative research group of agronomists, plant breeders, microbiologists, physiologists and molecularists to improve the symbiotic nitrogen fixation (SNF) and N 2 -dependent yield of common bean under moderate salinity in the Mediterranean basin are summarised. Agronomic surveys in reference production areas show large spatial and temporal variations in plant nodulation and growth, and in efficiency of utilisation of the rhizobial symbiosis. The latter was associated with a large rhizobial diversity, including new bean nodulating species. Macrosymbiont diversity in SNF and adaptation to NaCl was found. However, contrasts between plant genotypes could be altered by specific interactions with some native rhizobia. Therefore, variations in soil rhizobial population, in addition to agronomic practices and environmental constraints, may have contributed to erratic results observed in field inoculations. At the mechanistic level, nodule C and N metabolisms, and abcissic acid content, were related to SNF potential and tolerance to NaCl. Their relation with nodule conductance to O 2 diffusion was addressed by in situ hybridisation of candidate carbonic anhydrase and aquaporin genes in nodule cortex. The limits and prospects of the cooperative strategy are discussed. : S 0 1 6 8 -1 6 5 6 ( 0 1 ) 0 0 3 3 9 -X J.J. Dre6on et al. / Journal of Biotechnology 91 (2001) 257-268 258

Nitrogen-efficient and nitrogen-inefficient wheat genotypes were identified on the basis of the differential response of 16 wheat genotypes with low (1 mM) and high (25 mM) nitrogen (N) supply. Growth performance, measured in terms of fresh weight, dry weight and length of root and shoot, was higher in N-efficient than N-inefficient wheat genotypes at low N levels. Interestingly, although the growth of N-efficient genotypes did not show any change with increasing level of N supply, there was a marked increase in the growth of N-inefficient genotypes with the increase in N level. To work out the basis of this differential response of wheat genotypes to low N levels, we investigated the nitrate uptake rate of root and activities of nitrate assimilatory enzymes in the leaves of N-efficient and N-inefficient wheat genotypes. Nitrate uptake kinetics of these genotypes revealed that the uptake of nitrate in N-efficient genotypes was mediated by high-affinity nitrate transporter systems, whereas those of N-inefficient genotypes was mediated by low-affinity nitrate transporter systems. Study of the activities and expression levels of nitrate assimilatory enzymes in N-efficient and N-inefficient wheat genotypes showed that nitrate reductase (NR) and glutamine synthetase (GS) play important roles in N assimilation under low-nitrogen conditions.

The use of urea as an N fertilizer has increased to such an extent that it is now the most widely used fertilizer in the world. However, N losses as a result of ammonia volatilization lead to a decrease in its efficiency, therefore different methods have been developed over the years to reduce these losses. One of the most recent involves the use of urea combined with urease inhibitors, such as N-(n-butyl) thiophosphoric triamide (NBPT), in an attempt to delay the hydrolysis of urea in the soil. The aim of this study was to perform an in-depth analysis of the effect that NBPT use has on plant growth and N metabolism. Wheat plants were cultivated in a greenhouse experiment lasting 4 weeks and fertilized with urea and NBPT at different concentrations (0, 0.012, 0.062, 0.125%). Each treatment was replicated six times. A nonfertilized control was also cultivated. Several parameters related with N metabolism were analysed at the end of growth period. NBPT use was found to have visible effects, such as a transitory yellowing of the leaf tips, at the end of the first week of treatment. At a metabolic level, plants treated with the inhibitor were found to have more urea in their tissues and a lower amino acid content, lower glutamine synthetase activity, and lower urease and glutamine synthetase content at the end of the study period, whereas their urease activity seemed to have recovered by this stage.

The mammalian hepatic lobule is characterized by a Little is known about the alterations of metabolic ormarked functional heterogeneity. 1-3 According to their posiganization of the human liver tissue in chronic liver distion within the hepatic lobule, hepatocytes present signifieases. We therefore compared the distribution of the folcant differences in the level of many enzyme activities, such lowing zonal metabolic markers in 10 samples of normal as those involved in the carbohydrate, ammonia, lipid, and liver tissue, 10 samples of fibrotic tissue, and 22 samples xenobiotic metabolisms, 2-5 and in the expression of some seof cirrhotic tissue: (a) the enzymatic activities of glucosecreted products, such as plasma proteins. The metabolic or-6-phosphatase (G6P), lactate dehydrogenase (LDH), ganization of the mammalian hepatic lobule is well exemplisuccinate dehydrogenase (SDH), nicotinamide-adeninefied in the rat. In this species, gluconeogenesis, fatty acid dinucleotide-phosphate [NADPH] dehydrogenase (ND), oxydation, ureagenesis, and albumin synthesis predominate b-hydroxybutyrate dehydrogenase (HBDH), and glutamate dehydrogenase (GDH); (b) the protein glutamine in the periportal region of the hepatic lobule, while glycolysis, synthetase (GLS); and (c) albumin messenger RNA fatty acid synthesis, glutamine formation, and xenobiotic me-(mRNA). The normal human hepatic lobule was charactabolism predominate in the perivenous region. 7 The metaterized by the periportal predominance of G6P and SDH bolic zonation of the mammalian hepatic lobule is thought to enzymatic activities and albumin mRNAs, the periveplay an important physiological role by enabling the liver to nous predominance of ND and GDH, the restriction of simultaneously perform anabolic and catabolic activities, and GLS to a small perivenous compartment, and the preby allowing quick responses of the hepatic parenchyma to dominance of b-HBDH at the contact of both portal changes in the physiological status. 2,3,8 It is currently tracts and centrilobular veins. In fibrosis, the overall thought 2,3,8 that the maintenance of the metabolic organizametabolic organization of the normal liver tissue was tion of the liver depends mainly on microenvironmental facretained. The expression of periportal markers predomitors, including: (a) blood-borne factors, such as the gradients nated around enlarged portal tracts and that of perivein oxygen, substrates, and hormones determined by the uninous markers around residual centrilobular veins. GLS directional perfusion of the hepatic lobule by the portal blood was constantly detected at the contact of centrilobular flow, (b) pericellular factors, such as the zonal differences in veins. In cirrhotic nodules, no zonation was observed the pattern of innervation, the composition of the extracellufor most enzymatic activities or for albumin. Only G6P lar matrix, and the functional characteristics of nonparenchyusually predominated at the periphery of the nodules. mal liver cells; and possibly (c) paracrine interactions be-GLS was constantly undetectable. No difference actween hepatocytes of the different zones of the lobule.

This study demonstrated a general decrease in uptake of NH 4 + , urea and PO 4 3 − , activities of nitrogenase, glutamine synthetase, urease, alkaline phosphatase and ATPase following ultraviolet-B (UV-B) treatment and metal exposure, separately. As compared with an individual stressor the effect of their combination was more detrimental to the above processes. In contrast, a significant increase in NO 3 − uptake and nitrate reductase activity, following exposure of Anabaena doliolum to UV-B was observed. Kinetic study of all the processes demonstrated that the UV-B-induced structural change(s) in the enzymes/carriers could be responsible for uptake and assimilation of these nutrients. However, the metals seem to compete for the binding sites of the above enzymes/carriers. A synergistic interaction of UV-B and metals (Cu/Pb) seems to be due to an increased metal uptake by an altered membrane permeability, as a result of peroxidation of membrane lipids in UV-B exposed cells.

To study glutamine synthetase (GS) activity and glutamate uptake in the hippocampus and frontal cortex (FC) from rats with prehepatic portal vein hypertension. Male Wistar rats were divided into sham-operated group and a portal hypertension (PH) group with a regulated stricture of the portal vein. Animals were sacrificed by decapitation 14 d after portal vein stricture. GS activity was determined in the hippocampus and FC. Specific uptake of radiolabeled L-glutamate was studied using synaptosome-enriched fractions that were freshly prepared from both brain areas. We observed that the activity of GS increased in the hippocampus of PH rats, as compared to control animals, and decreased in the FC. A significant decrease in glutamate uptake was found in both brain areas, and was more marked in the hippocampus. The decrease in glutamate uptake might have been caused by a deficient transport function, significantly and persistent increase in this excitatory neurotransmitter activity. The ...

Genetic modiWcation of nitrogen metabolism via bacterial NADPH-dependent glutamate dehydrogenase (GDH; E.C.4.1.2.1) favorably alters growth and metabolism of C3 plants. The aim of this study was to examine the eVect of expression of GDH in the cytoplasmic compartment of Zea mays cells. The gdhA gene from Escherichia coli , that encoded a NADPH-GDH, was ligated to the ubiquitin promoter that incorporated the Wrst intron enhancer and used to transform Z. mays cv. 'H99' embryo cultures by biolistics. R0-R3 generations included selfed inbreds, back-crossed inbreds, and hybrids with B73 derivatives. The lines with the highest GDH speciWc activity produced infertile R0 plants. The highest speciWc activity of GDH from the fertile Z. mays plants was suYcient to alter phenotypes. Plant damage caused by the phosphinothricin in gluphosinate-type herbicides, glutamine synthetase (GS; EC 6.1.3.2) inhibitors, was less pronounced in Z. mays plants with gdhA pat than in gusA pat plants. Germination and grain biomass production were increased in gdhA transgenic plants in the Weld during seasons with signiWcant water deWcits but not over all locations. Water deWcit tolerance under controlled conditions was increased. Crops modiWed with gdhA may have value in semi-arid locations.

The tea-scented China roses largely correspond to the three recognized double-petaled Rosa odorata (Andrews) Sweet (Rosoideae, Rosaceae) varieties, which are the ancestors of modern hybrid tea roses and had a definite and permanent influence on the evolution of modern garden roses. Here the hypothesis of a hybrid origin of the tea-scented China roses between R. odorata var. gigantea and R. chinensis was tested. Two single-copy nuclear genes of the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and the chloroplast-expressed glutamine synthetase (ncpGS) together with two plastid loci (trnL-F and psbA-trnH) were sequenced for representative accessions of four R. odorata varieties, R. chinensis, and 28 other Rosa species. Phylogenetic relationships were estimated from two nuclear loci using maximum parsimony and Bayesian analyses, and a haplotype network was constructed on the combined plastid data using NETWORK. For GAPDH and ncpGS loci, the clonal sequences of the three double-petaled varieties were clustered into two clades, one clade with R. odorata var. gigantea, and the other with partial sequences of R. chinensis, which suggested that the tea-scented China roses were hybrids between R. odorata var. gigantea and R. chinensis. Two plastid loci suggested that R. odorata var. gigantea could be the maternal parent and R. chinensis the paternal parent.

A Tn5 insertional prototrophic mutant of Paracoccus denitrificans (UBM219) was generated which grew on high ( > 1 raM) but not low ( < 0.5 raM) ammonium as sole nitrogen source. It did not utilize nitrate and most amino acids except glutamate and aspartate. UBM219 showed more than 10-fold lower levels of a m m o n i u m (methylammonium) transport, aspartate and alanine aminotransferase, but more than 10-fold higher activities of glutamate dehydrogenase and glutamate synthase. This pleiotropy indicates a mutation in a regulatory gene affecting nitrogen metabolism in general.

Faba bean (Vicia faba L. var. minor cv. Alborea) and pea (Pisum sati6um L. cv. Lincoln) plants, inoculated with Rhizobium leguminosarum biovar. 6iciae strain GRA19, were treated with salt (100 mM NaCl) and/or nitrate (8 mM KNO 3 ) to test whether plants grown with inorganic-nitrogen are more tolerant to salinity than plants entirely reliant upon fixed nitrogen. According to the growth inhibition recorded, pea plants dependent on dinitrogen fixation proved more tolerant to salt stress than those N-fertilized, in contrast to results obtained for faba bean plants. This study therefore confirms that plants dependent on nitrogen fixation are not always more sensitive to salinity than are N-fertilized plants. Nitrate addition did not reduce the specific nitrogenase activity in pea, but did in faba bean. However, nodulation was inhibited in both legumes. The specific nitrogenase activity was more affected by salt treatment in N-fertilized plants for both legumes. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADH-GOGAT) was inhibited by salt stress both in N-fixing and in N-fertilized pea and faba bean plants.

The aim of the present study was to examine the effect of ultra-low-dose naloxone on pertussis toxin (PTX)-induced thermal hyperalgesia in rats and its underlying mechanisms. Male Wistar rats, implanted with an intrathecal catheter with or without a microdialysis probe, received a single intrathecal injection of PTX (1 mg in 5 ml saline). Four days after PTX injection, they were randomly given a different dose of naloxone (either 15 mg or 15 ng in 5 ml saline), followed by a morphine injection (10 mg in 5 ml saline) after 30 min. The results found that PTX injection induced thermal hyperalgesia and increasing excitatory amino acid (EAA; L-glutamate and L-aspartate) concentration in the spinal CSF dialysates. Ultra-low-dose naloxone not only preserved the antinociceptive effect of morphine but also suppressed the PTX-evoked EAA release as well. Moreover, ultra-low-dose naloxone plus morphine administration inhibited the downregulation of L-glutamate transporters (GTs) and the L-glutamate-metabolizing enzyme glutamine synthetase (GS), and, moreover, inhibited microglial activation and suppressed cytokine expression in PTX-treated rat spinal cords. These results show that ultra-low-dose naloxone preserves the antinociceptive effect of morphine in PTX-treated rats. The mechanisms include (a) inhibition of pro-inflammatory cytokine expression, (b) attenuation of PTX-evoked EAA release, and (c) reversion of the downregulation of GT expression.

Bacteria and fungi from pristine soil, never exposed to glufosinate herbicide, were isolated and analyzed for glufosinate tolerance. Seven of the 15 tested isolates were sensitive to 1 mM glufosinate (an active ingredient of many nonselective contact herbicides), 5 were resistant to 4 mM glufosinate and 3 even to 8 mM glufosinate in liquid medium. None of the isolated microorganisms carried the gene for glufosinate resistance bar (bialaphos resistance) in its genome and at least in some of glufosinate-resistant isolates the increased glutamine synthetase level was detected as a possible resistance mechanism. The transfer of the bar glufosinate resistance gene from transgenic maize Bt 176 into glufosinate-sensitive soil bacterium Bacillus pumilus S1 was not detected under the laboratory conditions by a classical plate count method and PCR. The ecological risk of potential bar gene transfer from genetically modified plants into soil microcosms under natural circumstances is discussed.

Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48 h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8 h of anaerobic treatment and, by immunoprecipitation of 35 S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/ glutamate synthase cycle play an important role in anaerobic amino acid accumulation.

Debaryomyces hansenii is adapted to grow in saline environments, accumulating high intracellular Na + concentrations. Determination of the DhGDH1encoded NADP-glutamate dehydrogenase enzymatic activity showed that it increased in a saline environment. Thus, it was proposed that, in order to overcome Na + inhibition of enzyme activity, this organism possessed salt-dependent mechanisms which resulted in increased activity of enzymes pertaining to the central metabolic pathways. However, the nature of the mechanisms involved in augmented enzyme activity were not analyzed. To address this matter, we studied the expression of DhGDH1 and DhGLN1 encoding glutamine synthetase, which constitute the central metabolic circuit involved in ammonium assimilation. It was found that: (1) expression of DhGDH1 is increased when D. hansenii is grown in the presence of high NaCl concentrations, while that of DhGLN1 is reduced, (2) DhGDH1 expression in Saccharomyces cerevisiae takes place in a GLN3-and HAP2,3-dependent manner and (3) salt-dependent DhGDH1 and DhGLN1 expression involves mechanisms which are limited to D. hansenii and are not present in S. cerevisiae. Thus, salt-dependent regulation of the genes involved in central metabolic pathways could form part of a strategy leading to the ability to grow under hypersaline conditions.

Metagenomics projects based on shotgun sequencing of populations of microorganisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium-and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

The effect of a hydrolyzed protein preparation on the growth and biomass yield of the blue-gree alga Spirulina platensis was studied. At the optimum dosage, the hydrolysate enhanced Spirulina productivity by almost 40% when compared to untreated cultures. This increased productivity correlated with an increase in the level of nitrogen assimilating enzymes, nitrate reductase and glutamine synthetase.