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Rod photoreceptors (PRs) use ribbon synapses to transmit visual information. To signal 'no light detected' they release glutamate continually to activate post-synaptic receptors. When light is detected glutamate release pauses. How a rod's individual ribbon enables this process was studied here by recording evoked changes in whole-cell membrane capacitance from wild-type and ribbonless (Ribeye-ko) mice. Wild-type rods filled with high (10 mM) or low (0.5 mM) concentrations of the Ca.sup.2+-buffer EGTA created a readily releasable pool (RRP) of 87 synaptic vesicles (SVs) that emptied as a single kinetic phase with a [tau]<0.4 ms. The lower concentration of EGTA accelerated Ca.sub.v channel opening and facilitated release kinetics. In contrast, ribbonless rods created a much smaller RRP of 22 SVs, and they lacked Ca.sub.v channel facilitation; however, Ca.sup.2+ channel-release coupling remained tight. These release deficits caused a sharp attenuation of rod-driven scotopic light responses. We conclude that the synaptic ribbon facilitates Ca.sup.2+-influx and establishes a large RRP of SVs.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells 1 – 4 . Macrophages and satellite cells communicate in different ways 1 – 5 , but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1 -knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles. Mouse models of muscle injuries and ageing characterized by low levels of intra-tissue glutamine are ameliorated by macrophage-specific deletion or systemic pharmacological inhibition of glutamate dehydrogenase 1, which results in constitutively high activity of glutamine synthetase.

The ALK inhibitor crizotinib as first-line therapy was associated with a significantly better response rate, longer progression-free survival, and greater improvement in quality of life measures than standard chemotherapy in patients with ALK -positive lung cancer. Rearrangements of the anaplastic lymphoma kinase ( ALK ) gene are present in 3 to 5% of non–small-cell lung cancers (NSCLCs). 1 , 2 They define a distinct subgroup of NSCLC that typically occurs in younger patients who have never smoked or have a history of light smoking and that has adenocarcinoma histologic characteristics. 3 – 5 Crizotinib is an oral small-molecule tyrosine kinase inhibitor of ALK, MET, and ROS1 kinases. 6 In phase 1 and 2 studies, crizotinib treatment resulted in objective tumor responses in approximately 60% of patients with ALK -positive NSCLC and in progression-free survival of 7 to 10 months. 7 – 9 In . . .

Nitrogen metabolism in citrus has received increased attention due to its effects on plant growth and productivity. However, little is known about the effects of nitrogen fertilization on nitrogen metabolism in young trees of citrus cultivar 'Huangguogan' (Citrus reticulata × Citrus sinensis). Here, genes encoding nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate dehydrogenase (GDH), and asparagine synthetase (AS), represented as HgNR, HgNiR, HgGS, HgGDH, and HgAS, respectively, were cloned from Huangguogan. Deduced protein sequences were analyzed and proteins were confirmed to be localized in their respective cellular organelles. Moreover, pot-cultured 'Huangguogan' seedlings were fertilized with 0 (N1), 1.36 (N2), 1.81 (N3), 2.26 (N4), or 2.72 (N5) kg N/year, for 12 months. Enzyme activity and enzyme-gene expression were studied in roots, leaves, and fruits at different stages. Finally, the effects of N application rate on root activity, leaf N, soluble protein, yield, and fruit quality at the ripening stage were measured. The results showed that: 1) HgNR, HgNiR, HgGDH, and HgAS gene products were found mainly in the cytoplasm and plasma membrane, while HgGS gene product was found mainly in cytoplasm and mitochondria. 2) Gene expression and enzyme activity differed among plant organs. As the root is in permanent direct contact with the soil we suggest that root gene expression and enzyme activity can be used as reference to determine N application rate. 3) Yield, fruit quality, enzyme activity, and enzyme-related gene expression were considerably lower at low than at high-N supply. However, they were all inhibited by excess nitrogen (i.e., 2.72 kg/year). Therefore, we recommend 1.81 kg N/year as the optimal N application rate for young 'Huangguogan' trees.

Glutamine synthetase, encoded by the gene GLUL , is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation. The enzyme glutamine synthetase is active in endothelial cell migration during angiogenesis, through autopalmitoylation and the regulation of RHOJ signalling.

Glutamate occupies a central position in amino acid metabolism in plants. The acidic amino acid is formed by the action of glutamate synthase, utilizing glutamine and 2-oxoglutarate. However, glutamate is also the substrate for the synthesis of glutamine from ammonia, catalysed by glutamine synthetase. The α-amino group of glutamate may be transferred to other amino acids by the action of a wide range of multispecific aminotransferases. In addition, both the carbon skeleton and α-amino group of glutamate form the basis for the synthesis of γ-aminobutyric acid, arginine, and proline. Finally, glutamate may be deaminated by glutamate dehydrogenase to form ammonia and 2-oxoglutarate. The possibility that the cellular concentrations of glutamate within the plant are homeostatically regulated by the combined action of these pathways is examined. Evidence that the well-known signalling properties of glutamate in animals may also extend to the plant kingdom is reviewed. The existence in plants of glutamate-activated ion channels and their possible relationship to the GLR gene family that is homologous to ionotropic glutamate receptors (iGluRs) in animals are discussed. Glutamate signalling is examined from an evolutionary perspective, and the roles it might play in plants, both in endogenous signalling pathways and in determining the capacity of the root to respond to sources of organic N in the soil, are considered.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism 1 . Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric G i . Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6–TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound G i can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6–TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation. Cryo-electron microscopy structures show that metabotropic glutamate receptor 2 forms a dimer to which only one G protein is coupled, revealing the basis for asymmetric signal transduction.

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings. Nerve cells or neurons communicate with each other by releasing specific molecules in the gap between them, the synapses. The sending neuron passes on messages through packets of chemicals called neurotransmitters, which are picked up by the receiving cell with the help of receptors on its surface. Neurons use different neurotransmitters to send different messages, but one of the most common ones is glutamate. There are two families of glutamate receptors: ionotropic receptors, which can open or close ion channels in response to neurotransmitters and control the transmission of a signal, and metabotropic receptors, which are linked to a specific protein and control the strength of signal. Our understanding of these two receptor families comes from animals with backbones, known as vertebrates. But the receptors themselves are ancient. We can trace the first family back as far as bacteria and the second back to single-celled organisms like amoebas. Vertebrates have six classes of ionotropic and three classes of metabotropic glutamate receptor. But other multi-celled animals also have these receptors, so this picture may not be complete. Here, Ramos-Vicente et al. mapped all major lineages of animals to reveal the evolutionary history of these receptors to find out if the receptor families became more complicated as brain power increased. The results showed that the glutamate receptors found in vertebrates are only a fraction of all the types that exist. In fact, before present-day animal groups emerged, the part of the genome that holds the ionotropic receptor genes duplicated three times. This formed four receptor subfamilies, and our ancestors had all of them. Across the animal kingdom, there are ten, not six, classes of ionotropic receptors and there is an extra class of metabotropic receptors. But only two subfamilies of ionotropic and three out of four metabotropic receptor classes are still present in vertebrates today. The current classification of glutamate receptors centers around vertebrates, ignoring other animals. But this new data could change that. A better knowledge of these new receptors could aid neuroscientists in better understanding the nervous system. And, using this technique to study other families of proteins could reveal more missing links in evolution.

Glutamate is an essential amino acid in both the energy and biosynthetic processes in plant cells. The aim of this work was to study changes in glutamate metabolism upon irradiation of maize (Zea mays L.) leaves with light of different spectral compositions, as well as to identify mechanisms regulating the work of enzymes involved in the studied process. A study was conducted of light-induced changes in glutamate metabolism in maize leaves, mediated by redirecting the glutamate flow to the γ-aminobutyric acid (GABA) shunt. Glutamate dehydrogenase (GDH) was more active in darkness, and the irradiation by red light inhibited the expression of both the Gdh1 and Gdh2 genes. EGTA and ruthenium red abolished the effects of light, indicating the participation of Ca2+ ions in phytochrome signal transduction. Contrary to GDH, glutamate decarboxylase (GAD) activity was moderately higher in the light, stimulated by red light, while far-red light reversed the effect. The effect of light on Gad expression was more pronounced than on GAD activity. Irradiation by red light also resulted in the increase in activity of GABA transaminase (GTA), which was abolished by far-red light. The third enzyme of the GABA shunt, succinic semialdehyde dehydrogenase (SSADH), was also activated by light. The effect of light on the expression of Ssadh1, but not on Ssadh2, was phytochrome-dependent. It is concluded that irradiation by light shifts glutamate metabolism from GDH to GAD with the activation of GABA transaminase and SSADH. This suggests that the GABA pathway plays a role in the maintenance of the tricarboxylic acid cycle in the light via bypassing its reactions when the 2-oxoglutarate dehydrogenase complex is inhibited and the cycle switches to the open mode.

Disulfide trapping and FRET studies define an agonist-induced conformational change in mGlu2 from inactive symmetric dimers with an interface at transmembrane domains (TMs) 4 and 5 to an active state with TM6s serving as the dimer interface. G protein–coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.