Transfer Of Glutamine Research Articles

Physiological synaptic activity and recognition memory require astroglial glutamine

Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.

Physiology and Morphological Correlates of Excitatory Transmission are Preserved in Glutamine Transporter SN1-Depleted Mouse Frontal Cortex

Glutamine is an astroglia-derived precursor of the neurotransmitter glutamate, and its astroglia-to-neuron transfer is controlled by distinct glutamine transporters on the astrocytic and neuronal sites. In this study, we focused on the role of astrocytic glutamine efflux-mediating system N transporter SN1 in the maintenance of glutamatergic neurotransmission by analyzing the electrophysiological parameters ex vivo in the brain slices from control mice and mice in which vivo-morpholino technique was used to diminish SN1 protein. The glutamatergic transmission was characterized by electrophysiological recordings, ultrastructure of neuron terminals, and determination of proteins related to glutamate synaptic transmission: synaptophysin, synaptotagmin, and vit1A. The space-restricted ∼51,5% reduction of SN1 protein did not affect the expression of the neuronal glutamine transporter SAT2. SN1 depletion resulted in a reduction of field potentials (FPs), unaltered frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs/mEPSCs), and presented a tendency towards a decrease of long-term potentiation (LTP). Ultrastructurally, preserved number of synaptic vesicles, primarily localized centrally of the cell body, correlates with unchanged levels of synaptic proteins. Collectively, the study indicates that glutamatergic transmission proceeds relatively independently of the SN1 - mediated glutamine transfer to the synapse.

Conditional Knockout of GLT-1 in Neurons Leads to Alterations in Aspartate Homeostasis and Synaptic Mitochondrial Metabolism in Striatum and Hippocampus.

Expression of the glutamate transporter GLT-1 in neurons has been shown to be important for synaptic mitochondrial function in the cerebral cortex. Here we determined whether neuronal GLT-1 plays a similar role in the hippocampus and striatum, using conditional GLT-1 knockout mice in which GLT-1 was inactivated in neurons by expression of synapsin-Cre (synGLT-1 KO). Ex vivo 13C-labelling using [1,2-13C]acetate, representing astrocytic metabolism, yielded increased [4,5-13C]glutamate levels, suggesting increased astrocyte-neuron glutamine transfer, in the striatum but not in the hippocampus of the synGLT-1 KO. Moreover, aspartate concentrations were reduced - 38% compared to controls in the hippocampus and the striatum of the synGLT-1 KO. Mitochondria isolated from the hippocampus of synGLT-1 KO mice exhibited a lower oxygen consumption rate in the presence of oligomycin A, indicative of a decreased proton leak across the mitochondrial membrane, whereas the ATP production rate was unchanged. Electron microscopy revealed reduced mitochondrial inter-cristae distance within excitatory synaptic terminals in the hippocampus and striatum of the synGLT-1 KO. Finally, dilution of 13C-labelling originating from [U-13C]glucose, caused by metabolism of unlabelled glutamate, was reduced in hippocampal synGLT-1 KO synaptosomes, suggesting that neuronal GLT-1 provides glutamate for synaptic tricarboxylic acid cycle metabolism. Collectively, these data demonstrate an important role of neuronal expression of GLT-1 in synaptic mitochondrial metabolism in the forebrain.

Placental transfer (clearance) of glutamine is dependent upon placental size in mice

Placental transfer (clearance) of glutamine is dependent upon placental size in mice

Adipocytes promote pancreatic cancer cell proliferation via glutamine transfer

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.

Introduction to the Glutamate-Glutamine Cycle.

The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain 14C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor. This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion but more recent research has seriously questioned this.This volume of Advances in Neurobiology is intended to provide a detailed discussion of recent developments in research aimed at delineating the functional roles of the cycle taking into account that in order for this system to work there must be a tight coupling between metabolism of glutamate in astrocytes, transfer of glutamine to neurons and de novo synthesis of glutamine in astrocytes. To understand this, knowledge about the activity and regulation of the enzymes and transporters involved in these processes is required and as can be seen from the table of contents these issues will be dealt with in detail in the individual chapters of the book.

No improvement of neuronal metabolism in the reperfusion phase with melatonin treatment after hypoxic-ischemic brain injury in the neonatal rat.

Mitochondrial impairment is a key feature underlying neonatal hypoxic-ischemic (HI) brain injury and melatonin is potentially neuroprotective through its effects on mitochondria. In this study, we have used (1) H and (13) C NMR spectroscopy after injection of [1-(13) C]glucose and [1,2-(13) C]acetate to examine neuronal and astrocytic metabolism in the early reperfusion phase after unilateral HI brain injury in 7-day-old rat pups, exploring the effects of HI on mitochondrial function and the potential protective effects of melatonin on brain metabolism. One hour after hypoxia-ischemia, astrocytic metabolism was recovered and glycolysis was normalized, whereas mitochondrial metabolism in neurons was clearly impaired. Pyruvate carboxylation was also lower in both hemispheres after HI. The transfer of glutamate from neurons to astrocytes was higher whereas the transfer of glutamine from astrocytes to neurons was lower 1h after HI in the contralateral hemisphere. Neuronal metabolism was equally affected in pups treated with melatonin (10mg/kg) immediately after HI as in vehicle treated pups indicating that the given dose of melatonin was not capable of protecting the neuronal mitochondria in this early phase after HI brain injury. However, any beneficial effects of melatonin might have been masked by modulatory effects of the solvent dimethyl sulfoxide on cerebral metabolism. Neuronal and astrocytic metabolism was examined by (13) C and (1) H NMR spectroscopy in the early reperfusion phase after unilateral hypoxic-ischemic brain injury and melatonin treatment in neonatal rats. One hour after hypoxia-ischemia astrocytic mitochondrial metabolism had recovered and glycolysis was normalized, whereas mitochondrial metabolism in neurons was impaired. Melatonin treatment did not show a protective effect on neuronal metabolism.

The anticonvulsant actions of carisbamate associate with alterations in astrocyte glutamine metabolism in the lithium-pilocarpine epilepsy model.

As reported previously, in the lithium-pilocarpine model of temporal lobe epilepsy (TLE), carisbamate (CRS) produces strong neuroprotection, leads to milder absence-like seizures, and prevents behavioral impairments in a subpopulation of rats. To understand the metabolic basis of these effects, here we injected 90mg/kg CRS or vehicle twice daily for 7days starting 1h after status epilepticus (SE) induction in rats. Two months later, we injected [1-13 C]glucose and [1,2-13 C]acetate followed by head microwave fixation after 15min. 13 C incorporation into metabolites was analyzed using 13 C magnetic resonance spectroscopy. We found that SE reduced neuronal mitochondrial metabolism in the absence but not in the presence of CRS. Reduction in glutamate level was prevented by CRS and aspartate levels were similar to controls only in rats displaying absence-like seizures after treatment [CRS-absence-like epilepsy (ALE)]. Glutamine levels in CRS-ALE rats were higher compared to controls in hippocampal formation and limbic structures while unchanged in rats displaying motor spontaneous recurrent seizures after treatment (CRS-TLE). Astrocytic mitochondrial metabolism was reduced in CRS-TLE, and either enhanced or unaffected in CRS-ALE rats, which did not affect the transfer of glutamine from astrocytes to neurons. In conclusion, CRS prevents reduction in neuronal mitochondrial metabolism but its effect on astrocytes is likely key in determining outcome of treatment in this model. To understand the metabolic basis of the strong neuroprotection and reduction in seizure severity caused by carisbamate (CRS) in the lithium-pilocarpine (Li-Pilo) model of temporal lobe epilepsy (TLE), we injected CRS for 7days starting 1h after status epilepticus and 2months later [1-13 C]glucose and [1,2-13 C]acetate. 13 C Magnetic resonance spectroscopy analysis was performed on brain extracts and we found that CRS prevented reduction in neuronal mitochondrial metabolism but its effect on astrocytes was likely key in determining outcome of treatment in this model. ALE=absence like epilepsy; acetyl CoA=acetyl coenzyme A; GS=glutamine synthetase; PAG=phosphate activated glutaminase; PC=pyruvate carboxylase; OAA=oxaloacetate; TCA cycle=tricarboxylic acid cycle.

The glutamine-glutamate/GABA cycle: function, regional differences in glutamate and GABA production and effects of interference with GABA metabolism.

The operation of a glutamine-glutamate/GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a well-known concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [(14)C]acetate or [(13)C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [(13)C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake.

Altered astrocyte-neuronal interactions after hypoxia-ischemia in the neonatal brain in female and male rats.

Increased susceptibility to excitotoxicity of the neonatal brain after hypoxia-ischemia (HI) may be caused by limited capacity of astrocytes for glutamate uptake, and mitochondrial failure probably plays a key role in the delayed injury cascade. Male infants have poorer outcome than females after HI, possibly linked to differential intermediary metabolism. [1-(13)C]glucose and [1,2-(13)C]acetate were injected at zero, 6, and 48 hours after unilateral HI in 7-day-old rats. Intermediary metabolism was analyzed with magnetic resonance spectroscopy. Mitochondrial metabolism was generally reduced in the ipsilateral hemisphere for ≤6 hours after HI, whereas contralaterally, it was reduced in neurons but not in astrocytes. Transfer of glutamate from neurons to astrocytes was increased in the contralateral, but not in the ipsilateral hemisphere at 0 hour, and reduced bilaterally at 6 hours after HI. The transfer of glutamine from astrocytes to glutamatergic neurons was unaltered in both hemispheres, whereas the transfer of glutamine to GABAergic neurons was increased ipsilaterally at 0 hour. Anaplerosis (astrocytes) was decreased, whereas partial pyruvate recycling (astrocytes) was increased directly after HI. Male pups had lower astrocytic mitochondrial metabolism than females immediately after HI, whereas that of females was reduced longer and encompassed both neurons and astrocytes. The prolonged depression in mitochondrial metabolism indicates that mitochondria are vulnerable targets in the delayed injury after neonatal HI. The degree of astrocytic malfunction may be a valid indicator of outcome after hypoxic/HI brain injury and may be linked to the differential outcome in males and females.

Book review: “Glial physiology and pathophysiology” by Alexei Verkhratsky and Arthur Butt, Wiley-Blackwell, 2013

Book review: “Glial physiology and pathophysiology” by Alexei Verkhratsky and Arthur Butt, Wiley-Blackwell, 2013

Neuron–Astrocyte Interactions, Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7day postnatal (P7) rats were injected with [1-(13)C]glucose and [1,2-(13)C]acetate and sacrificed 5, 10, 15, 30 and 45min later. Adult rats were injected and sacrificed after 15min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-(13)C]glucose and were sacrificed 30min later. Brain extracts were analysed using (1)H- and (13)C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-(13)C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-(13)C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-(13)C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.

Astrocytic energy metabolism and glutamate formation — relevance for 13C-NMR spectroscopy and importance of cytosolic/mitochondrial trafficking

Glutamate plays a double role in 13C-nuclear magnetic resonance (NMR) spectroscopic determination of glucose metabolism in the brain. Bidirectional exchange between initially unlabeled glutamate and labeled α-ketoglutarate, formed from pyruvate via pyruvate dehydrogenase (PDH), indicates the rate of energy metabolism in the tricarboxylic acid (V TCA) cycle in neurons (V PDH, n) and, with additional computation, also in astrocytes (V PDH, g), as confirmed using the astrocyte-specific substrate [ 13C]acetate. Formation of new molecules of glutamate during increased glutamatergic activity occurs only in astrocytes by combined pyruvate carboxylase (V PC) and astrocytic PDH activity. V PDH, g accounts for ∼15% of total pyruvate metabolism in the brain cortex, and V PC accounts for another ∼10%. Since both PDH-generated and PC-generated pyruvates are needed for glutamate synthesis, ∼20/25 (80%) of astrocytic pyruvate metabolism proceed via glutamate formation. Net transmitter glutamate [γ-aminobutyric acid (GABA)] formation requires transfer of newly synthesized α-ketoglutarate to the astrocytic cytosol, α-ketoglutarate transamination to glutamate, amidation to glutamine, glutamine transfer to neurons, its hydrolysis to glutamate and glutamate release (or GABA formation). Glutamate–glutamine cycling, measured as glutamine synthesis rate (V cycle), also transfers previously released glutamate/GABA to neurons after an initial astrocytic accumulation and measures predominantly glutamate signaling. An empirically established ∼1/1 ratio between glucose metabolism and V cycle may reflect glucose utilization associated with oxidation/reduction processes during glutamate production, which together with associated transamination processes are balanced by subsequent glutamate oxidation after cessation of increased signaling activity. Astrocytic glutamate formation and subsequent oxidative metabolism provide large amounts of adenosine triphosphate used for accumulation from extracellular clefts of neuronally released K + and glutamate and for cytosolic Ca 2+ homeostasis.

Reduced Astrocytic Contribution to the Turnover of Glutamate, Glutamine, and GABA Characterizes the Latent Phase in the Kainate Model of Temporal Lobe Epilepsy

The occurrence of spontaneous seizures in mesial temporal lobe epilepsy (MTLE) is preceded by a latent phase that provides a time window for identifying and treating patients at risk. However, a reliable biomarker of epileptogenesis has not been established and the underlying processes remain unclear. Growing evidence suggests that astrocytes contribute to an imbalance between excitation and inhibition in epilepsy. Here, astrocytic and neuronal neurotransmitter metabolism was analyzed in the latent phase of the kainate model of MTLE in an attempt to identify epileptogenic processes and potential biomarkers. Fourteen days after status epilepticus, [1-(13)C]glucose and [1,2-(13)C]acetate were injected and the hippocampal formation, entorhinal/piriform cortex, and neocortex were analyzed by (1)H and (13)C magnetic resonance spectroscopy. The (13)C enrichment in glutamate, glutamine, and γ-aminobutyric acid (GABA) from [1-(13)C]glucose was decreased in all areas. Decreased GABA content was specific for the hippocampal formation, together with a pronounced decrease in astrocyte-derived [1,2-(13)C]GABA and a decreased transfer of glutamine for the synthesis of GABA. Accumulation of branched-chain amino acids combined with decreased [4,5-(13)C]glutamate in hippocampal formation could signify decreased transamination via branched-chain aminotransferase in astrocytes. The results point to astrocytes as major players in the epileptogenic process, and (13)C enrichment of glutamate and GABA as potential biomarkers.

Synergistic action of hypoosmolarity and glutamine in inducing acute swelling of retinal glial (Müller) cells

High blood ammonia, elevated glutamine, and hyponatremia are pathogenic factors contributing to astrocytic swelling and brain edema in liver failure. We investigated the effects of hypoosmolarity, ammonia, and glutamine on the induction of glial cell swelling in freshly isolated slices of the rat retina. Glutamine, but not ammonia or hypoosmolarity per se, evoked a rapid (within one minute) swelling of retinal glial (Müller) cell bodies under hypoosmotic conditions. Under isoosmotic conditions, glutamine evoked a delayed swelling after 10 min of exposure. The effect of glutamine was concentration-dependent, with half-maximal and maximal effects at ∼ 0.1 and 0.5 mM. Glutamine in hypoosmotic solution induced a dissipation of the mitochondrial membrane potential. The effects on the mitochondrial membrane potential and the glial soma size were reduced by (i) agents which inhibit the transfer of glutamine into mitochondria and its hydrolysis there, (ii) inhibition of the mitochondrial permeability transition, (iii) inhibitors of oxidative-nitrosative stress, and (iv) inhibitors of phospholipase A(2) and cyclooxygenase. Glutamine-induced glial swelling was also prevented by ATP and adenosine, acting at adenosine A(1) receptors. The data suggest that hypoosmolarity accelerates the swelling-inducing effect of glutamine on retinal glial cells, and that swelling induction by glutamine is mediated by inducing oxidative-nitrosative stress, inflammatory lipid mediators, and mitochondrial dysfunction.

Alteration of glial‐neuronal metabolic interactions in a mouse model of Alexander disease

Alexander disease is a rare and usually fatal neurological disorder characterized by the abundant presence of protein aggregates in astrocytes. Most cases result from dominant missense de novo mutations in the gene encoding glial fibrillary acidic protein (GFAP), but how these mutations lead to aggregate formation and compromise function is not known. A transgenic mouse line (Tg73.7) over-expressing human GFAP produces astrocytic aggregates indistinguishable from those seen in the human disease, making them a model of this disorder. To investigate possible metabolic changes associated with Alexander disease Tg73.7 mice and controls were injected simultaneously with [1-(13)C]glucose to analyze neuronal metabolism and [1,2-(13)C]acetate to monitor astrocytic metabolism. Brain extracts were analyzed by (1)H magnetic resonance spectroscopy (MRS) to quantify amounts of several key metabolites, and by (13)C MRS to analyze amino acid neurotransmitter metabolism. In the cerebral cortex, reduced utilization of [1,2-(13)C]acetate was observed for synthesis of glutamine, glutamate, and GABA, and the concentration of the marker for neuronal mitochondrial metabolism, N-acetylaspartate (NAA) was decreased. This indicates impaired astrocytic and neuronal metabolism and decreased transfer of glutamine from astrocytes to neurons compared with control mice. In the cerebellum, glutamine and GABA content and labeling from [1-(13)C]glucose were increased. Evidence for brain edema was found in the increased amount of water and of the osmoregulators myo-inositol and taurine. It can be concluded that astrocyte-neuronal interactions were altered differently in distinct regions.

A novel back-door pathway for glutamine release from GlnBP revealed by steered molecular dynamics simulation

The Glutamine transfer system consists of three components: glutamine binding protein (GlnBP), the transmembrane receptor (TMR) and the ATP binding subunit. It is typical of periplasmic transfer systems in Escherichia coli and belongs to the ABC (ATP-binding cassettes) super family. Until now, the mechanism of glutamine release from its receptor remains unknown, also little information is available on the interactions between the substrate-bound complex and the TMR because of a lack of structure information of the TMR. In our study, a steered molecular dynamics (SMD) method is used to explore the possible pathway for glutamine release from its receptor GlnBP. It is found that a novel back-door pathway, rather than a front-door pathway is more reasonable. This work may help understand the substrate release mechanism of the periplasmic transfer system and additionally give some clues as to how the substrate-bound complex binds to its TMR and completes the subsequent translocation of the substrate.

Excitatory Synaptic Transmission Persists Independently of the Glutamate–Glutamine Cycle

The glutamate-glutamine cycle is thought to be integral in continuously replenishing the neurotransmitter pool of glutamate. Inhibiting glial transfer of glutamine to neurons leads to rapid impairment in physiological and behavioral function; however, the degree to which excitatory synaptic transmission relies on the normal operation of this cycle is unknown. In slices and cultured neurons from rat hippocampus, we enhanced the transfer of glutamine to neurons, a fundamental step in this cycle, by adding exogenous glutamine. Although raising glutamine augments synaptic transmission by increasing vesicular glutamate, access to this synthetic pathway by exogenously applied glutamine to neurons is delayed and slow, challenging mechanisms linking the rapid effects of pharmacological inhibitors to decreased vesicular glutamate. We find that pharmacological inhibitors of glutamine synthetase or system A transporters cause an acute depression of basal synaptic transmission that is rapidly reversible, which is unlikely to be attributable to the rapid loss of vesicular glutamate. Furthermore, release of vesicular glutamate remains robust even during the prolonged removal of glutamine from pure neuronal cultures. We conclude that neurons have the capacity to store or produce glutamate for long periods of time, independently of glia and the glutamate-glutamine cycle.

Snapshots of tRNA sulphuration via an adenylated intermediate

Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.

Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-1β on rat chondrocytes

Objective: To determine whether overexpression of glutamine: fructose-6-phosphate amidotransferase (GFAT) in synoviocytes will antagonize the response to interleukin-1β (IL-1β) of chondrocytes and synovial fibroblasts in co-culture. Methods: Synovial fibroblasts from the rat were transduced by an adenovirus carrying the cDNA for GFAT and then co-cultured with rat chondrocytes encapsulated in alginate beads. Following challenge with 1, 5, or 10 ng/ml of IL-1β for 24 h, proteoglycan synthesis by the chondrocytes was determined by incorporation of Na 2 35SO 4. Production of nitric oxide (NO) and prostaglandin E 2(PGE 2) were monitored by assay of conditioned medium from the co-culture. Results: IL-1β treatment of untransduced-synoviocyte/chondrocyte co-cultures resulted in markedly decreased proteoglycan synthesis by the chondrocytes, and increased NO and PGE 2levels in the culture medium. In contrast, adenovirus-mediated transfer of GFAT in synoviocytes prevented both the decrease in chondrocyte proteoglycan synthesis and increases in NO and PGE 2provoked by IL-1β. Conclusions: Our study suggests that in a synoviocyte/chondrocyte co-culture system, overexpression of GFAT by synoviocytes significantly inhibits subsequent stimulation by IL-1β in vitro. Since GFAT is the rate limiting enzyme in the synthesis of intracellular glucosamine and its derivatives, these results may open new possibilities for osteoarthritis treatment.