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In eukaryotic translation initiation, the 48S preinitiation complex (PIC) scans the 5' untranslated region of mRNAs to search for the cognate start codon (AUG) with assistance from various eukaryotic initiation factors (eIFs). Cognate start codon recognition is precise, rejecting near-cognate codons with a single base difference. However, the structural basis of discrimination of near-cognate start codons was not known. We have captured multiple yeast 48S PICs with a near-cognate AUC codon at the P-site, revealing that the AUC codon induces instability in the codon-anticodon at the P-site, leading to a disordered N-terminal tail of eIF1A. Following eIF1 dissociation, the N-terminal domain of eIF5 fails to occupy the vacant eIF1 position, and eIF2β becomes flexible. Consequently, 48S with an AUC codon is less favourable for initiation. Furthermore, we observe hitherto unreported metastable states of the eIF2-GTP-Met-tRNAMet ternary complex, where the eIF2β helix-turn-helix domain may facilitate eIF5 association by preventing eIF1 rebinding to 48S PIC. Finally, a swivelled head conformation of 48S PIC appears crucial for discriminating incorrect and selection of the correct codon-anticodon pair during translation initiation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness., Competing Interests: NO authors have competing interests.

The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The bifunctional homooligomeric enzyme Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) and its encoding gene ALDH18A1 were associated with disease in 1998. Two siblings who presented paradoxical hyperammonemia (alleviated by protein), mental disability, short stature, cataracts, cutis laxa, and joint laxity, were found to carry biallelic ALDH18A1 mutations. They showed biochemical indications of decreased ornithine/proline synthesis, agreeing with the role of P5CS in the biosynthesis of these amino acids. Of 32 patients reported with this neurocutaneous syndrome, 21 familial ones hosted homozygous or compound heterozygous ALDH18A1 mutations, while 11 sporadic ones carried de novo heterozygous ALDH18A1 mutations. In 2015 to 2016, an upper motor neuron syndrome (spastic paraparesis/paraplegia SPG9) complicated with some traits of the neurocutaneous syndrome, although without report of cutis laxa, joint laxity, or herniae, was associated with monoallelic or biallelic ALDH18A1 mutations with, respectively, dominant and recessive inheritance. Of 50 SPG9 patients reported, 14 and 36 (34/2 familial/sporadic) carried, respectively, biallelic and monoallelic mutations. Thus, two neurocutaneous syndromes (recessive and dominant cutis laxa 3, abbreviated ARCL3A and ADCL3, respectively) and two SPG9 syndromes (recessive SPG9B and dominant SPG9A) are caused by essentially different spectra of ALDH18A1 mutations. On the bases of the clinical data (including our own prior patients' reports), the ALDH18A1 mutations spectra, and our knowledge on the P5CS protein, we conclude that the four syndromes share the same pathogenic mechanisms based on decreased P5CS function. Thus, these syndromes represent a continuum of increasing severity (SPG9A < SPG9B < ADCL3 ≤ ARCL3A) of the same disease, P5CS deficiency, in which the dominant mutations cause loss-of-function by dominant-negative mechanisms., (© 2020 SSIEM.)

Objectives: Since the introduction of endovascular treatment for cerebral aneurysms, hospitals in which subarachnoid hemorrhage is treated show different availability and/or preferences towards both treatment modalities. The main aim is to evaluate the clinical and angiographic results according to the hospital's treatment preferences applied., Methods: This study was conducted based on use of the subarachnoid hemorrhage database of the Vascular Pathology Group of the Spanish Neurosurgery Society. Centers were classified into 3 subtypes according to an index in the relationship between endovascular and surgical treatment as: endovascular preference, high endovascular preference, and elevated surgical preference. The clinical results and angiographic results were evaluated among the 3 treatment strategies., Results: From November 2004 to December 2017, 4282 subarachnoid hemorrhage patients were selected for the study: 630 (14.7%) patients from centers with surgical preference, 2766 (64.6%) from centers with endovascular preference, and 886 (20.7%) from centers with high endovascular preference. The surgical preference group obtained the best angiographic results associated with a greater complete exclusion (odds ratio: 1.359; 95% confidence interval: 1.025-1.801; P = 0.033). The surgical preference subgroup obtained the best outcome at discharge (65.45%), followed by the high endovascular preference group (61.5%) and the endovascular preference group (57.8%) (odds ratio: 1.359; 95% confidence interval: 1.025-1.801; P = 0.033)., Conclusions: In Spain, there is significant variability in aneurysm exclusion treatment in aneurysmal subarachnoid hemorrhage. Surgical centers offer better results for both surgical and endovascular patients. A multidisciplinary approach and the maintenance of an elevated quality of surgical competence could be responsible for these results., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The paper 'Interaction of N-acetyl-l-glutamate kinase with the PII signal transducer in the non-photosynthetic alga Polytomella parva: Co-evolution towards a hetero-oligomeric enzyme' by Selim et al. highlights how the study of a true taxonomic oddity, the heterotrophic unicellular alga P. parva, has been instrumental in uncovering the large potential for adaptive variation in the signaling complex of PII with the enzyme N-acetylglutamate kinase (NAGK). This complex modifies the regulatory properties of NAGK, allowing nitrogen stockpiling as arginine. In P. parva, a stable PII-NAGK complex is formed which lacks regulation by canonical PII effectors but which exhibits novel adaptive responses to nitrogen abundance mediated by glutamine, a neo-effector of PII proteins of photosynthetic eukaryotes., (© 2019 Federation of European Biochemical Societies.)

Introduction: Transsphenoidal surgical removal is the preferred treatment of most pituitary adenomas. Postoperative cerebrospinal fluid (CSF) leakage is the leading cause of morbidity after this procedure, with an incidence rate that varies from 0,5-15% in the main published series., Objectives: The primary objective of this study was to establish the incidence of postoperative CSF leakage in a sample of surgeries performed at the University Hospital of La Ribera by the same surgical team. The secondary objectives were to: ascertain the distinctive features between patients with and without postoperative CSF leakage, identify risk factors for their development, evaluate the relationship between the surgical technique for closing the sella turcica and the onset of postoperative CSF leakage and evaluate different treatment regimens for this complication., Methods: The data of 302 consecutive transsphenoidal surgical procedures for pituitary adenoma removal which were performed between 1999 and 2017 were retrospectively reviewed., Results and Conclusions: The incidence of postoperative CSF leakage in our series was 2,3% (in accordance with similar published studies). It was possible to correlate intraoperative CSF leakage with two variables: pituitary macroadenoma and tumors with suprasellar extension (P<.005). This correlation did not exist for postoperative CSF leakage. We found a statistically significant correlation between intraoperative and postoperative CSF leakage (P<.005). Due to the low incidence of postoperative CSF leakage in our series, it was not possible to identify risk factors for its development., (Copyright © 2019 Sociedad Española de Neurocirugía. Publicado por Elsevier España, S.L.U. All rights reserved.)

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNA i .

In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNA i in a 'P IN ' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNA i . Substitutions of eIF5 residues involved in the eIF5-NTD/tRNA i interaction influenced initiation at near-cognate UUG codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection., Competing Interests: JL, TH, AS, JN, SK, YG, RK, JL, VR No competing interests declared, AH Reviewing editor, eLife

P II , a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. P II sensing is mediated by the P II allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of P II function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a P II regulatory network. Studies on these organisms yielded a first structure of a P II complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how P II can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which P II can control gene expression. A small adaptor protein, PipX, is sequestered by P II when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of P II -NAGK, P II -PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally P II regulatory functions and reveal the changing shapes and interactions of the T-loops of P II depending on the partner and on the allosteric effectors bound to P II . Cyanobacterial studies have also revealed that in the P II -PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this P II -PipX regulatory network and of K D values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs.

In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Arterial supply and venous drainage at the foramen magnum is variable. Two main forms of clinical presentation, intracranial and spinal, can be differentiated when a dural arteriovenous fistula (DAVF) is found at this level. We describe a case of a 68-year-old patient with a progressive paraparesis, diagnosed of dural arteriovenous fistula located at the posterior lip of foramen magnum. We review, in this setting, the vascular radiological anatomy of those fistulas and its important correlation with neurologic clinical symptoms., (Copyright © 2016 Sociedad Española de Neurocirugía. Published by Elsevier España. All rights reserved.)

Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

Background: Fluorescence-guided microsurgical resections of high-grade gliomas using 5-aminolevulinic acid (5-ALA) is superior to conventional microsurgery. An optical device, usually a modified microscope, is needed for these procedures. However, an exoscope may be implemented for fluorescence techniques. We present the use of an exoscope to perform tumor resection guided by 5-ALA fluorescence in 21 consecutive patients with high-grade glioma and two neuronavigation-guided biopsies., Methods: Twenty-three patients underwent operations. Tumor volume and localization were quantified with pre- and postoperative volumetric MRI in non-biopsy cases., Results: In non-biopsy cases, the age range was 20 to 79 years, with a median of 56 (interquartile range = 45-66). Histological analysis indicated that 14 had glioblastoma multiforme, 2 grade-III oligodendrogliomas and 1 anaplastic astrocytoma, 3 metastases and 1 low-grade astrocytoma. Total resection was achieved in 15 cases; subtotal resection was performed in 5 patients. The result was partial resection in one case. There was no perioperative mortality. The median fluorescence intensity, on a scale of 1-5, was 4.5 in the GBM group (IQR = 4-5), 3 (IQR = 2.5-3.5) in anaplastic glioma, and 2.5 (IQR = 2.25-2.75) for oligodendrogliomas. Of the three metastases, one showed fluorescence level 4. As for the two biopsy cases, one was anaplastic astrocytoma and one glioblastoma multiforme. The samples obtained were fluorescent in both cases., Conclusions: An exoscope can be also used for fluorescence-guided surgery with 5-aminolevulinic acid (5-ALA) and neuronavigation-guided biopsy. With an important advantage of low cost, this allows the surgeon to perform collaborative surgeries and adds agility to the procedure.

Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo-electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site.

Background: The introduction of fluorescence-guided resection allows a better identification of tumor tissue and its more radical resection. We describe our experience with a modified exoscope to detect 5 ALA-induced fluorescence in neuronavigation-guided brain surgery or biopsy of malignant brain tumors., Methods: Thirty-eight patients with a suspected preoperative diagnosis of high-grade astrocytoma were included. We used a neuronavigation device and a high-definition exoscope system with a built-in filter to detect 5-ALA fluorescence in all cases. Thirty patients underwent craniotomy with tumor resection and 8 underwent frameless stereotactic brain biopsy., Results: Histopathological diagnosis confirmed the presence of high-grade gliomas in 34 patients. Total resection was achieved in 23 cases and subtotal in 7. No relevant complications related to the administration of 5-ALA were detected., Conclusions: The use of the exoscope in 5-ALA fluorescence-guided tumor surgery has twofold implications: during brain tumor surgery it can be considered a valuable tool to achieve a more radical resection of the lesion, and when applied to a biopsy of a suspected brain high-grade glioma, it decreases the possibility of a negative biopsy.

PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

When nitrogen is abundant, prokaryotic and eukaryotic oxygen-producing photosynthetic organisms store nitrogen as arginine, by relieving feedback inhibition of the arginine biosynthesis controlling enzyme, N-acetylglutamate kinase (NAGK). The signalling protein PII, an ancient and widely distributed nitrogen/carbon/ADP/ATP sensor, mediates feedback inhibition relief of NAGK by binding to this enzyme. PII phosphorylation or PII binding of ADP or 2-oxoglutarate prevents PII-NAGK complex formation. Crystal structures of NAGK, cyanobacterial and plant PII and corresponding PII-NAGK complexes have been recently determined. In these complexes, two polar PII trimers sandwich one ring-like NAGK hexamer. Each PII subunit contacts one NAGK subunit, triggering a symmetry-restricted narrowing of the NAGK ring, with concomitant adoption by the arginine sites of a low-affinity conformation.

Photosynthetic organisms can store nitrogen by synthesizing arginine, and, therefore, feedback inhibition of arginine synthesis must be relieved in these organisms when nitrogen is abundant. This relief is accomplished by the binding of the PII signal transduction protein to acetylglutamate kinase (NAGK), the controlling enzyme of arginine synthesis. Here, we describe the crystal structure of the complex between NAGK and PII of Synechococcus elongatus, at 2.75-A resolution. We prove the physiological relevance of the observed interactions by site-directed mutagenesis and functional studies. The complex consists of two polar PII trimers sandwiching one ring-like hexameric NAGK (a trimer of dimers) with the threefold axes of these molecules aligned. The binding of PII favors a narrow ring conformation of the NAGK hexamer that is associated with arginine sites having low affinity for this inhibitor. Each PII subunit contacts one NAGK subunit only. The contacts map in the inner circumference of the NAGK ring and involve two surfaces of the PII subunit. One surface is on the PII body and interacts with the C-domain of the NAGK subunit, helping widen the arginine site found on the other side of this domain. The other surface is at the distal region of a protruding large loop (T-loop) that presents a novel compact shape. This loop is inserted in the interdomain crevice of the NAGK subunit, contacting mainly the N-domain, and playing key roles in anchoring PII on NAGK, in activating NAGK, and in complex formation regulation by MgATP, ADP, 2-oxoglutarate, and by phosphorylation of serine-49.

Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Angstroms-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K(i) = 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase. The inhibition of NAGK by arginine was found to be pH independent and to depend sigmoidally on the concentration of arginine, with a Hill coefficient (N) of approximately 4, and the 50% inhibitory arginine concentration (I0.5) was shown to increase with temperature, approaching above 65 degrees C the I0.50 observed at 37 degrees C with the mesophilic NAGK of Pseudomonas aeruginosa (the best-studied arginine-inhibitable NAGK). At 75 degrees C, the inhibition by arginine of T. maritima NAGK was due to a large increase in the Km for acetylglutamate triggered by the inhibitor, but at 37 degrees C arginine also substantially decreased the Vmax of the enzyme. The NAGKs of T. maritima and P. aeruginosa behaved in gel filtration as hexamers, justifying the sigmoidicity and high Hill coefficient of arginine inhibition, and arginine or the substrates failed to disaggregate these enzymes. In contrast, Escherichia coli NAGK is not inhibited by arginine and is dimeric, and thus the hexameric architecture may be an important determinant of arginine sensitivity. Potential thermostability determinants of T. maritima NAGK are also discussed.

Previous studies of our group showed that C1-C2 spinal cord stimulation increases carotid and brain blood flow in normal conditions in the goat and dog and it has a beneficial vasomotor effect in a model of vasospasm in the rat. For further clinical application it seemed rational to investigate the possible vascular changes mediated by this technique in experimental brain infarction. To this aim, 45 New Zealand rabbits were used. Brain infarction was produced by bilateral carotid ligation in 15, unilateral microcoagulation of the middle cerebral artery in 15 and by microcoagulation of the vertebral artery at the craniocervical junction in the other 15. One week later, following daily clinical scoring and cortical and posterior fossa blood flow readings by laser Doppler, a period of 120 min of right C1-C2 spinal cord electric stimulation was performed. A mean of 27% increase in previous blood flow recordings was obtained at the right hemisphere and a mean of 32% in the posterior fossa. This procedure was used in 10 patients presenting with various cerebral low perfusion syndromes. Though not constant, an increase in alertness, retention, speech, emotional lability and performance in skilled acts was achieved. No MR changes were observed, though SPECT readings showed an increase in blood flow in the penumbral perilesional area.