Inhibition Of Glutamine Synthetase Activity Research Articles

Abundant urinary amino acids activate glutamine synthetase-encoding glnA by two different mechanisms in Escherichia coli.

Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.IMPORTANCEKnowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections-most often caused by Escherichia coli-growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.

Effects of zinc toxicity on the nitrogen-fixing cyanobacterium Anabaena sphaerica-ultastructural, physiological and biochemical analyses.

The current study describes the mechanisms of zinc toxicity in the cyanobacterium Anabaena sphaerica after eight days treatment with 10 mg L-1 ZnCl2. The application of zinc not only showed elevated accumulation of the metal inside the cells but also exhibited devastating impacts on the cell numbers, morphology, and ultrastructure of A. sphaerica. The effects of zinc on the pigments contents, oxygen evolution rate, Fv/Fm, electron transport rate, and carbohydrate content were also evaluated in A. sphaerica. Moreover, zinc adversely affected nutrient uptake and the cellular energy budget in the test cyanobacterium which in turn hampered heterocyst development and nitrogen fixation. Alongside, the cyanobacterium experienced zinc-mediated non-competitive inhibition of glutamine synthetase activity, curtailed synthesis of amino acids and proteins. Furthermore, drastically reduced total lipid and increased unsaturated lipid contents were also the prominent characteristics of zinc stressed A. sphaerica. Most importantly, zinc stress caused severe damages to the protein, lipid, and DNA by triggering hydrogen peroxide generation and accumulation of oxidized glutathione. Therefore, excess zinc is highly toxic to the cyanobacterium A. sphaerica, and the mechanisms of its toxicity followed a cascade of events including oxidative stress mediated geopardisation of growth and ultrastructure, metabolic derangements, and macromolecular damages.

Regulation of glutamine synthetase activity by transcriptional and posttranslational modifications negatively influences ganoderic acid biosynthesis in Ganoderma lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.

Superfluous glutamine synthetase activity in Chinese Hamster Ovary cells selected under glutamine limitation is growth limiting in glutamine-replete conditions and can be inhibited by serine.

Passaging and expansion of animal cells in lean maintenance medium could result in periods of limitation of some nutrients. Over time, such stresses could possibly result in selection of cells with metabolic changes and contribute to heterogeneity. Here, we investigate whether selection of Chinese Hamster Ovary (CHO) cells under glutamine limitation results in changes in growth under glutamine-replete conditions. In glutamine-limiting medium, compared to control cells passaged in glutamine-rich medium, the selected cells showed higher glutamine synthetase (GS) activity and attained a higher peak viable cell density (PVCD). Surprisingly, in glutamine-replete conditions, selected cells still showed a higher GS activity but a lower PVCD. We show that in glutamine-replete medium, PVCD of selected cells was restored on (a) inhibition of GS activity with methionine sulfoximine, (b) supplementation of aspartate-without affecting GS activity, and (c) supplementation of serine, which is reported to inhibit GS in vitro. Consistent with the reported effect of serine, inhibition of GS activity was observed upon serine supplementation along with reduced growth of cells under glutamine-limiting conditions. The latter observation is important for the design of glutamine-free culture medium and feed used for GS-CHO and GS-NS0. In summary, we show that CHO cells selected under glutamine limitation have superfluous GS activity in glutamine-replete medium, which negatively affects their PVCD. This may be due to its effect on availability of aspartate which was the limiting nutrient for the growth of selected cells in glutamine-replete conditions.

Hypoxic Preconditioning Maintains GLT-1 Against Transient Global Cerebral Ischemia Through Upregulating Cx43 and Inhibiting c-Src.

Transient global cerebral ischemia (tGCI) causes excessive release of glutamate from neurons. Astrocytic glutamate transporter-1 (GLT-1) and glutamine synthetase (GS) together play a predominant role in maintaining glutamate at normal extracellular concentrations. Though our previous studies reported the alleviation of tGCI-induced neuronal death by hypoxic preconditioning (HPC) in hippocampal Cornu Ammonis 1 (CA1) of adult rats, the underlying mechanism has not yet been fully elaborated. In this study, we aimed to investigate the roles of GLT-1 and GS in the neuroprotection mediated by HPC against tGCI and to ascertain whether these roles can be regulated by connexin 43 (Cx43) and cellular-Src (c-Src) activity. We found that HPC decreased the level of extracellular glutamate in CA1 after tGCI via maintenance of GLT-1 expression and GS activity. Inhibition of GLT-1 expression with dihydrokainate (DHK) or inhibition of GS activity with methionine sulfoximine (MSO) abolished the neuroprotection induced by HPC. Also, HPC markedly upregulated Cx43 and inhibited p-c-Src expression in CA1 after tGCI, whereas inhibition of Cx43 with Gap26 dramatically reversed this effect. Furthermore, inhibition of p-c-Src with 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo (3, 4-d) pyrimidine (PP2) decreased c-Src activity, increased protein levels of GLT-1 and Cx43, enhanced GS activity, and thus reduced extracellular glutamate level in CA1 after tGCI. Collectively, our data demonstrated that reduced extracellular glutamate induced by HPC against tGCI through preventing the reduction of GLT-1 expression and maintaining GS activity in hippocampal CA1, which was mediated by upregulating Cx43 expression and inhibiting c-Src activity.

Glutamine Synthetase in Endothelial Cells of the Blood–brain Barrier: Less But Not Insignificant?

Background and Aim: The liver plays a major role in regulating ammonia levels in the blood. Therefore, during liver disease, the loss of hepatic function leads to hyperammonemia and consequently increased brain ammonia and hepatic encephalopathy (HE). Reducing ammonia neurotoxicity through systemic ammonia-lowering strategies remains the mainstay therapeutic strategy for HE. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which during the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in the endothelial cells of the BBB. Methods: Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS activity and protein (brain lysate was used as positive control). GS was also evaluated by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, GS activity was assessed in ECs exposed to 1 mM of ammonium chloride for 48 h. Finally, GS activity and protein expression were evaluated in ECs submitted to plasma from 6-week bile-duct ligated (BDL) rats or sham-operated controls for 72 h. Results: ECs expressed mRNA, protein and activity of GS. However, EC's expression (normalized to g/protein) of GS was lower compared to brain lysate control samples (P < 0.05). GS protein expression in CMV showed similar results to that of brain but GS activity was significantly less in CMV (P < 0.05). Using immunohistochemistry, GS was detected in ECs and in CMV from naïve rats. When ECs were exposed to ammonia (1 mM), an increase in GS activity was demonstrated (P < 0.05). However, when exposed to conditioned medium from BDL rats, GS activity and protein expression were decreased when compared to sham-operated controls (P < 0.05). Conclusion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme in CMVs compared to brain, could reason why GS has never been reported in these cells. Interestingly, ammonia stimulates GS activity in ECs, but GS activity is decreased following treatment with plasma from BDL rats. This suggests other factors such as oxidative stress and inflammation, could inhibit GS activity. We speculate that a downregulation of GS in the BBB allows for a faster entry of ammonia into the brain and therefore may play a significant role in the onset of HE. We anticipate upregulating GS in ECs of the BBB could become a new therapeutic target for HE. The authors have none to declare.

Glutamate metabolism of astrocytes during hyperbaric oxygen exposure and its effects on central nervous system oxygen toxicity.

Hyperbaric oxygen (HBO) has been used widely in many underwater missions and clinical work. However, exposure to extremely high oxygen pressure may cause central nervous system oxygen toxicity (CNS-OT). The regulation of astrocyte glutamate metabolism is closely related to epilepsy. This study aimed to observe the effects of HBO exposure on glutamate metabolism in astrocytes and confirm the role of glutamate metabolism in CNS-OT. Anesthetized rats were exposed to 5 atmosphere absolute HBO for 80 min and microdialysis samples of brain interstitial fluid were continuously collected. Extracellular glutamate and glutamine concentrations were also detected. Freely moving rats were exposed to HBO of the same pressure for 20 min and glutamine synthetase (GS) activity in brain tissues was measured. Finally, we observed the effects of different doses of drugs related to glutamate metabolism on the latency of CNS-OT. Results showed that HBO exposure significantly increased glutamate content, whereas glutamine content was significantly reduced. Moreover, HBO exposure significantly reduced GS activity. Glutamate transporter-1 (GLT-1) selective antagonist ceftriaxone prolonged CNS-OT latency, whereas GLT-1 selective inhibitor dihydrokainate shortened CNS-OT latency. In summary, HBO exposure improved glutamate concentration and reduced glutamine concentration by inhibition of GS activity. GLT-1 activation also participated in the prevention of HBO-induced CNS-OT. Our research will provide a potential new target to terminate or attenuate CNS-OT.

Effect of Liver Damage and Hyperbaric Oxygenation on Glutamine Synthetase of Hepatocytes.

Activity of glutamine synthetase in the hepatocytes of healthy animals and animals with chronic CCl4-induced hepatitis was studied on white mature female rats after liver resection (15-20% of organ weight) and hyperbaric oxygenation (3 atm, 50 min, 3 times). Surgically operated left and non-operated middle lobes of the liver were analyzed on day 3 after liver resection and exposure to hyperbaric oxygenation. On day 65 of CCl4 poisoning, activity of glutamine synthetase decreased in both lobes and did not recover on day 3 after toxin cessation. Liver resection under conditions of CCl4-induced hepatitis restored reduced activity of glutamine synthetase in both liver lobes to the normal level. In healthy rats, the increase in glutamine synthetase activity after liver resection was found only in the middle lobe of the liver. Hyperbaric oxygenation enhanced the stimulatory effect of liver resection on glutamine synthetase activity in hepatocytes during chronic CCl4-induced hepatitis. In healthy animals with liver resection, activity of glutamine synthetase did not change after hyperbaric oxygenation, while normally oxygenation inhibited glutamine synthetase activity.

In vivo and In vitro neurochemical-based assessments of wastewater effluents from the Maumee River area of concern

Wastewater treatment plant (WWTP) effluents contain potentially neuroactive chemicals though few methods are available to screen for the presence of such agents. Here, two parallel approaches (in vivo and in vitro) were used to assess WWTP exposure-related changes to neurochemistry. First, fathead minnows (FHM, Pimephales promelas) were caged for four days along a WWTP discharge zone into the Maumee River (Ohio, USA). Grab water samples were collected and extracts obtained for the detection of alkylphenols, bisphenol A (BPA) and steroid hormones. Second, the extracts were then used as a source of in vitro exposure to brain tissues from FHM and four additional species relevant to the Great Lakes ecosystem (rainbow trout (RT), river otter (RO), bald eagle (BE) and human (HU)). The ability of the wastewater (in vivo) or extracts (in vitro) to interact with enzymes (monoamine oxidase (MAO) and glutamine synthetase (GS)) and receptors (dopamine (D2) and N-methyl-d-aspartate receptor (NMDA)) involved in dopamine and glutamate-dependent neurotransmission were examined on brain homogenates. In vivo exposure of FHM led to significant decreases of NMDA receptor binding in females (24–42%), and increases of MAO activity in males (2.8- to 3.2-fold). In vitro, alkylphenol-targeted extracts significantly inhibited D2 (66% in FHM) and NMDA (24–54% in HU and RT) receptor binding, and induced MAO activity in RT, RO, and BE brains. Steroid hormone-targeted extracts inhibited GS activity in all species except FHM. BPA-targeted extracts caused a MAO inhibition in FHM, RT and BE brains. Using both in vivo and in vitro approaches, this study shows that WWTP effluents contain agents that can interact with neurochemicals important in reproduction and other neurological functions. Additional work is needed to better resolve in vitro to in vivo extrapolations (IVIVE) as well as cross-species differences.

Inhibition of Astrocytic Glutamine Synthetase by Lead is Associated with a Slowed Clearance of Hydrogen Peroxide by the Glutathione System

Lead intoxication in humans is characterized by cognitive impairments, particularly in the domain of memory, where evidence indicates that glutamatergic neurotransmission may be impacted. Animal and cell culture studies have shown that lead decreases the expression and activity of glutamine synthetase (GS) in astrocytes, yet the basis of this effect is uncertain. To investigate the mechanism responsible, the present study exposed primary astrocyte cultures to a range of concentrations of lead acetate (0–330 μM) for up to 24 h. GS activity was significantly reduced in cells following 24 h incubation with 100 or 330 μM lead acetate. However, no reduction in GS activity was detected when astrocytic lysates were co-incubated with lead acetate, suggesting that the mechanism is not due to a direct interaction and involves intact cells. Since GS is highly sensitive to oxidative stress, the capacity of lead to inhibit the clearance of hydrogen peroxide (H2O2) was investigated. It was found that exposure to lead significantly diminished the capacity of astrocytes to degrade H2O2, and that this was due to a reduction in the effectiveness of the glutathione system, rather than to catalase. These results suggest that the inhibition of GS activity in lead poisoning is a consequence of slowed H2O2 clearance, and supports the glutathione pathway as a primary therapeutic target.

Mercury-induced neurotoxicity and neuroprotective effects of berberine.

Mercury-induced neurotoxicity and neuroprotective effects of berberine.

Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine—relevance to the treatment of neurological diseases

At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.

Response of Arabidopsis thaliana, Seedlings to Cadmium in Relation to Ammonium Availability

This study explored the natural variability of Arabidopsis thaliana to find out its response to ammonium availability and characterize the tolerance capacity to cadmic stress under low, average or high nitrogen supplies. Growth was determined by different morphological traits and metabolic enzymes. Plant growth was inhibited by cadmium at low nitrogen regime. But, at average or high nitrogen conditions, plant growth seems to be no affected by cadmium. Cadmium inhibited glutamine synthetase activity and stimulated glutamate dehydrogenase one in order to tolerate cadmium stress. Cadmium enhanced proline and sugar tenor in average or high nitrogen plants parallel with activation of Rubisco. Observations show that cadmium-induced stress was alleviated by optimal ammonium availability.

Inhibition of Glutamine Synthetase by Phosphinothricin Leads to Transcriptome Reprograming in Root Nodules of Medicago truncatula

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

Effects of Acutely Elevated Hydrostatic Pressure in a Rat Ex Vivo Retinal Preparation

A new experimental glaucoma model was developed by using an ex vivo rat retinal preparation to examine the effects of elevated hydrostatic pressure on retinal morphology and glutamine synthetase (GS) activity. Ex vivo rat retinas were exposed to elevated hydrostatic pressure for 24 hours in the presence of glutamate or glutamate receptor antagonists and examined histologically. GS activity was assessed by colorimetric assay. Pressure elevation induced axonal swelling in the nerve fiber layer. Axonal swelling was prevented by a combination of non-N-methyl-d-aspartate (non-NMDA) receptor antagonist and an NMDA receptor antagonist, indicating that the damage results from activation of both types of glutamate receptor. When glial function was preserved, the typical changes induced by glutamate consisted of reversible Müller cell swelling resulting from excessive glial glutamate uptake. The irreversible Müller cell swelling in hyperbaric conditions may indicate that pressure disrupts glutamate metabolism. Indeed, elevated pressure inhibited GS activity. In addition, glutamate exposure after termination of pressure exposure exhibited apparent Müller cell swelling. These results suggest that the neural degeneration observed during pressure elevation is caused by impaired glial glutamate metabolism after uptake.

The food-associated fungal neurotoxin ochratoxin A inhibits the absorption of glutamate by astrocytes through a decrease in cell surface expression of the excitatory amino-acid transporters GLAST and GLT-1

The food-associated mycotoxin ochratoxin A (OTA) has been demonstrated to be deleterious to numerous cell types including brain cells. Although OTA has been proved to be toxic to astrocytes, no other investigation has been conducted on the impact of OTA on astrocytic functions. In the present study, we evaluated the effect of OTA on one of the major astrocytic functions, i.e. the reabsorption of extracellular glutamate. We found that OTA suppressed glutamate absorption by rat cortical astrocytes with a half inhibitory concentration of 1.3 and 10.1 μM in the absence and presence of fetal calf serum. Although OTA inhibits glutamine synthetase activity, this effect was not involved in OTA-mediated alteration of glutamate absorption since decrease in enzyme activity only occurred at high cytotoxic concentrations of toxin (100 μM). Similarly, alterations in the expression of the excitatory amino-acid transporters were not involved since OTA failed to modify total expression level of GLAST and GLT-1. We found that inhibition of glutamate absorption by OTA was due to a decrease in the expression of GLAST and GLT-1 at the cell surface. We propose that, in addition to being directly toxic to neurons and astrocytes, OTA could also cause the death of brain cells through inhibition of glutamate uptake by astrocytes, leading to the accumulation of extracellular glutamate and ultimately to excitotoxicity.

Dual regulation of Ca2+‐dependent glutamate release from astrocytes: Vesicular glutamate transporters and cytosolic glutamate levels

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation-induced Ca2+-dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca2+-dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca2+ increase. Inhibition of glutamine synthetase activity by L-methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca2+-dependent glutamate release, without affecting intracellular Ca2+ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca2+-dependent release of glutamate from astrocytes.

Wnt and steroid pathways control glutamate signalling by regulating glutamine synthetase activity in osteoblastic cells

Glutamate signalling has recently been found functional also outside the central nervous system, especially in bone. Glutamate is converted to glutamine by glutamine synthetase (GS), which is therefore able to regulate intracellular concentrations of glutamate. We previously characterized the induction of GS expression by glucocorticoids (GCs) in human osteoblast-like cells. Besides this observation, the mechanisms controlling GS in bone are unknown. Therefore, the aim of our present study was to investigate further the regulation of GS in osteoblastic cells. We observed that vitamin D inhibited basal and, even more efficiently, GC-stimulated GS activity by affecting both the mRNA and protein levels of the enzyme in human MG-63 osteoblast-like cells. In osteoblasts derived from rat bone marrow stem cells (rMSCs), GS activity was induced accordingly by the osteogenic culture conditions including GCs. Also in these primary cells, vitamin D clearly inhibited GS activity. In addition, the canonical Wnt signalling pathway was characterized as a negative regulator of GS activity. All these changes in GS activity were reflected on the intracellular glutamate concentration. Our results provide novel evidence that GS activity and expression are regulated by several different signalling pathways in osteoblastic cells. Therefore, GS is a strategic enzyme in controlling glutamate concentration in bone environment: GCs decreased the amount of this signalling molecule while vitamin D and Wnt signalling pathway increased it. Interestingly, GS activity and expression declined rapidly when the rMSC derived osteoblasts began to mineralize. Due to its downregulation during osteoblast mineralization, GS could be held as a marker for osteoblast development. Further supporting this, GS activity was stimulated and intracellular glutamate concentration maintained by the N-methyl-d-aspartate (NMDA) type glutamate receptor antagonist MK801, which inhibited osteogenic differentiation of the rMSCs. GS, a novel target for both steroidal and Wnt pathways in bone, might be a central player in the regulation of osteoblastogenesis and/or intercellular signal transmission. Therefore, the proper understanding of the interplay of these three signalling cascades, i.e., steroidal, Wnt, and glutamate signalling, gives vital information on how bone cells communicate together aiming to keep bone healthy.

Protein phosphatase PphA from Synechocystis sp. PCC 6803: the physiological framework of PII-P dephosphorylation

The phosphorylated signal transduction protein P(II) (P(II)-P) in the cyanobacterium Synechocystis sp. strain PCC 6803 is dephosphorylated by PphA, a protein phosphatase of the 2C family (PP2C). In this study, the physiological conditions of P(II)-P dephosphorylation were investigated with respect to the in vivo specificity of P(II)-P towards PphA and the cellular abundance of PphA in cells growing under different nitrogen regimes. Furthermore, the consequences of impaired P(II)-P dephosphorylation with respect to short-term inhibition of glutamine synthetase (GS) were studied. With a contribution of approximately 15 % of total Mn(2+)-dependent p-nitrophenyl phosphate hydrolysis activity, PphA has only a minor impact on the total PP2C activity in Synechocystis extracts. Nevertheless, residual P(II)-P dephosphorylation in PphA-deficient cells could only be observed after prolonged incubation in the presence of ammonium. The abundance of PphA correlates with the phosphorylation state of P(II) under nitrogen-replete conditions and is specifically enhanced by nitrite. Regulation of pphA expression operates at the post-transcriptional level. In the presence of nitrate/nitrite, PphA is present in molar excess over P(II)-P, enabling the cells to rapidly dephosphorylate P(II)-P in response to changing environmental conditions. A PphA-deficient mutant is not impaired in short-term inhibition of GS activity following ammonium treatment. Down-regulation of GS occurs by induction of gif genes (encoding GS inactivating factors 7 and 17), which is controlled by NtcA-mediated gene repression. Thus, impaired P(II)-P dephosphorylation does not affect ammonium-prompted inactivation of NtcA.

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (Vi) was almost completely inhibited in the presence of MSX, suggesting that Vi is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (Vs) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.