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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.

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.

Conventional imaging using CT and bone scan has insufficient sensitivity when staging men with high-risk localised prostate cancer. We aimed to investigate whether novel imaging using prostate-specific membrane antigen (PSMA) PET-CT might improve accuracy and affect management. In this multicentre, two-arm, randomised study, we recruited men with biopsy-proven prostate cancer and high-risk features at ten hospitals in Australia. Patients were randomly assigned to conventional imaging with CT and bone scanning or gallium-68 PSMA-11 PET-CT. First-line imaging was done within 21 days following randomisation. Patients crossed over unless three or more distant metastases were identified. The primary outcome was accuracy of first-line imaging for identifying either pelvic nodal or distant-metastatic disease defined by the receiver-operating curve using a predefined reference-standard including histopathology, imaging, and biochemistry at 6-month follow-up. This trial is registered with the Australian New Zealand Clinical Trials Registry, ANZCTR12617000005358. From March 22, 2017 to Nov 02, 2018, 339 men were assessed for eligibility and 302 men were randomly assigned. 152 (50%) men were randomly assigned to conventional imaging and 150 (50%) to PSMA PET-CT. Of 295 (98%) men with follow-up, 87 (30%) had pelvic nodal or distant metastatic disease. PSMA PET-CT had a 27% (95% CI 23–31) greater accuracy than that of conventional imaging (92% [88–95] vs 65% [60–69]; p<0·0001). We found a lower sensitivity (38% [24–52] vs 85% [74–96]) and specificity (91% [85–97] vs 98% [95–100]) for conventional imaging compared with PSMA PET-CT. Subgroup analyses also showed the superiority of PSMA PET-CT (area under the curve of the receiver operating characteristic curve 91% vs 59% [32% absolute difference; 28–35] for patients with pelvic nodal metastases, and 95% vs 74% [22% absolute difference; 18–26] for patients with distant metastases). First-line conventional imaging conferred management change less frequently (23 [15%] men [10–22] vs 41 [28%] men [21–36]; p=0·008) and had more equivocal findings (23% [17–31] vs 7% [4–13]) than PSMA PET-CT did. Radiation exposure was 10·9 mSv (95% CI 9·8–12·0) higher for conventional imaging than for PSMA PET-CT (19·2 mSv vs 8·4 mSv; p<0·001). We found high reporter agreement for PSMA PET-CT (κ=0·87 for nodal and κ=0·88 for distant metastases). In patients who underwent second-line image, management change occurred in seven (5%) of 136 patients following conventional imaging, and in 39 (27%) of 146 following PSMA PET-CT. PSMA PET-CT is a suitable replacement for conventional imaging, providing superior accuracy, to the combined findings of CT and bone scanning. Movember and Prostate Cancer Foundation of Australia. [Display omitted]

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.

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.

This trial assessed alum-formulated glutamic acid decarboxylase, the 65-kD isoform (GAD65), a major autoantigen in type 1 diabetes. In patients with recent-onset disease; the compound did not significantly alter the loss of C peptide or improve clinical outcomes. The clinical onset of type 1 diabetes is manifested by the effects of inadequate insulin secretion due to the immunologic destruction of pancreatic-islet beta cells. 1 Despite replacement therapy with exogenous insulin, type 1 diabetes is associated with substantial morbidity and mortality. 2 , 3 Even modest preservation of insulin secretion appears to reduce short- and long-term complications of type 1 diabetes. 4 – 9 Initial attempts at immunosuppression to treat type 1 diabetes had a positive effect but one that was outweighed by treatment-related adverse events. 10 – 12 More recently, selective immunosuppression has been attempted. Phase 2 trials showed promising efficacy, but phase 3 studies . . .

Group I metabotropic glutamate receptors (mGlu 1 and mGlu 5 ) are promising targets for multiple psychiatric and neurodegenerative disorders. Understanding the subtype selectivity of mGlu 1 and mGlu 5 allosteric sites is essential for the rational design of novel modulators with single- or dual-target mechanism of action. In this study, starting from the deposited mGlu 1 and mGlu 5 crystal structures, we utilized computational modeling approaches integrating docking, molecular dynamics simulation, and efficient post-trajectory analysis to reveal the subtype-selective mechanism of mGlu 1 and mGlu 5 to 10 diverse drug scaffolds representing known negative allosteric modulators (NAMs) in the literature. The results of modeling identified six pairs of non-conserved residues and four pairs of conserved ones as critical features to distinguish the selective NAMs binding to the corresponding receptors. In addition, nine pairs of residues are beneficial to the development of novel dual-target NAMs of group I metabotropic glutamate receptors. Furthermore, the binding modes of a reported dual-target NAM (VU0467558) in mGlu 1 and mGlu 5 were predicted to verify the identified residues that play key roles in the receptor selectivity and the dual-target binding. The results of this study can guide rational structure-based design of novel NAMs, and the approach can be generally applicable to characterize the features of selectivity for other G-protein-coupled receptors.

This study aimed to test whether injections of alum-formulated glutamic acid decarboxylase 65 (GAD), a major autoantigen in type 1 diabetes mellitus, would reverse recent-onset disease. C-peptide levels declined in both the treatment group and the control group, without significant between-group differences at month 15 (the primary end point), but they had declined significantly more slowly with treatment by month 30. The authors conclude that alum-formulated GAD may help preserve residual insulin secretion in patients with recent-onset type 1 diabetes. Study patients received injections of alum-formulated glutamic acid decarboxylase 65 (GAD). C-peptide levels declined in both the treatment group and the control group, but they had declined significantly more slowly with treatment by month 30. Type 1 diabetes mellitus is an autoimmune disease 1 that causes substantial morbidity and mortality. 2 , 3 Even modest residual insulin secretion, with stimulated C-peptide levels above 0.2 nmol per liter (0.6 ng per milliliter), has been reported to provide clinically meaningful benefits in terms of reducing long-term complications. 4 However, most attempts to preserve residual beta-cell function have achieved minimal benefits or have been associated with adverse effects. 5 – 14 Treatment with anti-CD3 monoclonal antibodies appears promising, although many patients in whom this approach has been used have had therapy-related adverse events. 15 , 16 As an alternative to immunosuppression, autoantigens may be used to . . .