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The α2adrenergic receptors (α2ARs) are G protein-coupled receptors (GPCRs) that respond to adrenaline and noradrenaline and couple to the Gi/o family of G proteins. α2ARs play important roles in regulating the sympathetic nervous system. Dexmedetomidine is a highly selective α2AR agonist used in post-operative patients as an anxiety-reducing, sedative medicine that decreases the requirement for opioids. As is typical for selective αAR agonists, dexmedetomidine consists of an imidazole ring and a substituted benzene moiety lacking polar groups, which is in contrast to βAR-selective agonists, which share an ethanolamine group and an aromatic system with polar, hydrogen-bonding substituents. To better understand the structural basis for the selectivity and efficacy of adrenergic agonists, we determined the structure of the α2BAR in complex with dexmedetomidine and Go at a resolution of 2.9 Å by single-particle cryo-EM. The structure reveals the mechanism of α2AR-selective activation and provides insights into Gi/o coupling specificity.

Human muscarinic receptor M2is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand–residue contacts in M2receptors compared to M3receptors leads to subtype selectivity of AF-DX 384.

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR–Gicomplex to previously determined structures of other GPCRs bound to the stimulatory G protein Gsreveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Giprotein-coupling specificity of the µOR. A cryo-electron structure of the µ-opioid receptor in complex with the peptide agonist DAMGO and the inhibitory G protein Gireveals structural determinants of its G protein-binding specificity.

The molecular mechanisms underlying the protein assembly at synaptic junctions are thought to be important for neural functions. PSD-95, one of the major postsynaptic density proteins, is composed of three PDZ domains (PDZ1, PDZ2, and PDZ3), an SH3 domain, and a GK (guanylate kinase ) domain. It binds to theN-methyl-d-aspartate glutamate receptor NR2 subunit or to the Shaker-type K+channel, Kv1.4, via the PDZ1 or PDZ2 domain, whereas PDZ3 binds to distinct partners. The intramolecular interaction of these multiple domains has been implicated in efficient protein clustering. We introduced missense and deletion mutations into PDZ1 (PDZ1mΔ) and/or PDZ2 (PDZ2mΔ) of the full-length PSD-95 to disrupt the association of each domain with the target proteins, while preserving the overall structure. The ion channel clustering activities of the PSD-95 mutants were analyzed in COS-1 cells coexpressing each mutant and Kv1.4. The mutant bearing the dysfunctional PDZ2 (PSD-95:1-2mΔ) showed significantly reduced clustering efficiency, whereas the mutant with the dysfunctional PDZ1 (PSD-95:1mΔ-2) exhibited activity comparable with the wild-type activity. Furthermore, we also examined the requirements for the position of PDZ2 in full-length PSD-95 by constructing a series of PDZ1-PDZ2 inversion mutants. Surprisingly, the clustering activity of PSD-95:2-1mΔ was severely defective. Taken together, these findings show that PDZ2, which is endowed with the highest affinity for Kv1.4, is required for efficient ligand binding. In addition, the ligand binding at the position of the second PDZ domain in full-length PSD-95 is prerequisite for efficient and typical cluster formation. This study suggests that the correct placement of the multiple domains in the full-length PSD-95 protein is necessary for the optimal protein activity.

Abstract: We treated a Japanese man with aneurysmal dilatation of the bovine graft transplanted for femorofemoral bypass. The reconstruction was performed successfully by the replacement with a Dacron graft 29 months after the first operation. Aneurysmal dilatation was observed over the entire length of the graft, and the wall of the graft was fragile. Disruption of collagen fibers and bleeding into the graft wall from the arteries which had been newly formed in abundance were extensive. The histological findings suggested that bovine grafts should be used for only a short period or for the replacement of small arteries.

Correspondence to: Kiyohito Yamamoto, M.D., Department of Thoracic and Cardiovascular Surgery, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-02, Japan--> Abstract. Ten cases with iliac artery disease were evaluated using rotational angiography with revolution of the arm of the X-ray tube support unit. The tip of the catheter was advanced to the infrarenal aorta, and contrast medium was injected at a rate of 6–10 ml/second. Digital angiographic images from multiple projection angles were acquired at 15 frames/second with a rotation speed at about 10°/second and a range of greater than 90°. This method made it possible to detect ulcerations of the artery, which could not be visualized using conventional single-plane angiography. The three-dimensional structure of a dissection could also be visualized. In seven cases of occlusive iliac artery disease, comparison with rotational angiography revealed that anteroposterior views of single-plane angiography underestimated the extent of the disease in four cases and overestimated the extent of the disease in one case. In two cases, no additional clinical information was obtained. In conclusion, rotational angiography provides accurate three-dimensional information concerning the lumen of the iliac arteries.

Fresh tissue slices were fixed in 5% formalin containing 0.9% NaCl for 10-20 min and frozen sections therefrom floated for 3 hr at 37°C on an incubating mixture made as follows. Sodium pyrophosphate (Na4P2O7-12H2O), 1.088 gm was dissolved in 20-30 ml of distilled water and to this was added ferric chloride (FeCl3-6H2O), 0.61 gm dissolved in 10-15 ml of water. The precipitate was just dissolved by cautiously adding 5-10% aqueous Na2CO3 solution and the pH adjusted to 7.2 with 1N HCl. The volume was made up to 100 ml and 0.9 gm of NaCl added. Before use, 1 ml of 10% Mg(NO3) was added. After incubation, sections were washed 10-15 min in 0.9% NaCl, then mounted on glass slides and air-dried. When dry, the slides were immersed in 0.9% NaCl containing 0.2-0.5% ammonium sulfide for 2-3 min, then dehydrated rapidly through graded alcohols, cleared, and covered in balsam. Sites of pyrophosphatase activity stained in various shades of green. Acid pyrophosphatase also was histochemically demonstrated by the same principle, excepting that the substrate solution was adjusted to pH 3.7-4.0 with acetate buffer. The pattern of distribution of pyrophosphatase and glycerophosphatase was almost identical.

A taste-modifying protein, curculin, has been crystallized by the vapor diffusion method using polyethylene glycol 400 as a precipitant. The crystals belong to orthorhombic space group P2₁2₁2₁ with unit cell dimensions: a=105 Å, b=271 Å, c=48·7 Å. The crystals diffract X-rays to at least a resolution of 3.0 Å and are suitable for X-ray crystallographic studies. Copyright 1994, 1999 Academic Press