The metabotropic glutamate receptors (mGluRs) are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. Here we have determined three different crystal structures of the extracellular ligand-binding region of mGluR1--in a complex with glutamate and in two unliganded forms. They all showed disulphide-linked homodimers, whose 'active' and 'resting' conformations are modulated through the dimeric interface by a packed alpha-helical structure. The bi-lobed protomer architectures flexibly change their domain arrangements to form an 'open' or 'closed' conformation. The structures imply that glutamate binding stabilizes both the 'active' dimer and the 'closed' protomer in dynamic equilibrium. Movements of the four domains in the dimer are likely to affect the separation of the transmembrane and intracellular regions, and thereby activate the receptor. This scheme in the initial receptor activation could be applied generally to G-protein-coupled neurotransmitter receptors that possess extracellular ligand-binding sites.

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC 50 ) of quisqualate for the soluble receptor was 3.8 ؋ 10 ؊8 M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate > > ibotenate > L-glutamate Ϸ (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.Glutamate receptors are divided into two distinct classes: ionotropic glutamate receptors (iGluRs) 1 and metabotropic glutamate receptors (mGluRs) (1, 2). The iGluRs consist of Nmethyl-D-aspartate receptors and non-N-methyl-D-aspartate receptors. Non-N-methyl-D-aspartate receptors are further subdivided into two groups: ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and kainate receptors. iGluRs are ligand-gated ion channels that transduce glutamate binding into cation influx. mGluRs that have been discovered most recently comprise eight subtypes, which are divided into three groups according to agonist selectivity, coupling to different effector systems, and sequence homology (3-6). Group I includes mGluR1 and mGluR5, which are coupled to inositol phospholipid metabolism. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) are negatively coupled to adenylate cyclase activity. Functional analyses of these mGluRs are now avidly being performed. The evidence is accumulating that mGluRs modulate excitatory synaptic transmission (7) through various neural transduction pathways, such as regulation of neurotransmitter release (8), influences on ion channel activity (9), and modulation of synaptic plasticity (10).mGluRs have a remarkably large extracellular domain that has no homology with the other G protein-coupled receptors (GPCRs) except Ca 2ϩ -sensing receptors (11). Previous experiments (12, 13) have proposed that the ligand binding site resides mainly in t...

One form of communication that is common in all cultures is people singing together. Singing together reflects an index of cognitive synchronization and cooperation of human brains. Little is known about the neural synchronization mechanism, however. Here, we examined how two brains make one synchronized behavior using cooperated singing/humming between two people and hyperscanning, a new brain scanning technique. Hyperscanning allowed us to observe dynamic cooperation between interacting participants. We used functional near-infrared spectroscopy (fNIRS) to simultaneously record the brain activity of two people while they cooperatively sang or hummed a song in face-to-face (FtF) or face-to-wall (FtW) conditions. By calculating the inter-brain wavelet transform coherence between two interacting brains, we found a significant increase in the neural synchronization of the left inferior frontal cortex (IFC) for cooperative singing or humming regardless of FtF or FtW compared with singing or humming alone. On the other hand, the right IFC showed an increase in neural synchronization for humming only, possibly due to more dependence on musical processing.

Expression of several members of the Bcl-2 family proteins was investigated by means of both immunohistochemical analysis in 30 invasive ductal adenocarcinomas and 23 intraductal papillary-mucinous tumors (IPMTs) and immunoblot analysis in 6 cancer tissues and 7 pancreatic cancer cell lines. We found that Bcl-2 was expressed in 23%, Bax in 53%, Bcl-X in 90%, and Mcl-1 in 90% of the invasive ductal adenocarcinomas. In intraductal papillary-mucinous adenocarcinomas, the expression rate of Bax was 44% and those of Bcl-XL and Mcl-1 were 88%; these values were higher than those for intraductal papillary-mucinous adenomas. Immunoblot analysis identified Bcl-XL as the predominant form of the Bcl-X protein in both pancreatic cancer tissues and cell lines, and demonstrated that both Bcl-XL and Mcl-1 protein levels were uniformly high in all cell lines. These results suggest that an imbalance between antiapoptosis proteins (such as Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic proteins (such as Bax and Bcl-Xs) is involved in the distinctive biologic features of adenocarcinomas of the pancreas. Furthermore, predominantly high expressions of Bcl-XL and Mcl-1 in intraductal papillary-mucinous adenocarcinomas might be involved in the carcinogenesis in IPMT of the pancreas.

Inula helenium (Compositae) is a widely occurring perennial herb in Europe and East Asia. Its roots have been traditionally used as a diaphoresis and a diuretic expectorant agent in Europe, as a fragrance agent for home medicines in Japan, 1) and as agents of tuberculotic enterorrhea, chronic enterogastritis and bronchitis and a preservative in China. 2)Native Americans used infusion and decoctions of this roots to treat lung disorders and against tuberculosis.3) The investigation of the genus Inula has shown the sesquiterpene lactone and essential oil groups, [4][5][6] and some phenolic acids and flavonoids were evaluated as other constituents of this genus. 7,8) Many sesquiterpenes were isolated from Inula helenium.9-11) The principal ingredient, alantolactone has strong anthelmintic and antibacterial activities. 9)As part of our screening for antiproliferative constituents in natural resources, we examined the roots of I. helenium grown in Tibet. The MeOH extract of the roots of this plant showed antiproliferative activities against three tumor cell lines: human gastric adenocarcinoma cells (MK-1), human uterus carcinoma (HeLa) and mouse melanoma (B16F10) cells.The MeOH extract was partitioned with n-hexane, CHCl 3 , EtOAc, and then n-BuOH. The n-hexane fraction showed strong antiproliferative activity against MK-1, HeLa and B16F10 cells, while the EtOAc and n-BuOH fractions had very low activity as shown in Fig. 1. The n-hexane fraction was fractionated in the manner described in the Materials and Methods section, and seven sesquiterpenes were isolated. The antiproliferative activities of the fractions and seven isolated compounds against MK-1, HeLa, and B16F10 cell lines will be discussed. MATERIALS AND METHODSMaterial Dried roots were obtained from Mr. Hiroyuki Kaito of Soma Co., 7-9-1 Tanimati, Tyuo-ku, Osaka 542, Japan on December 7, 1999. A voucher specimen (KPU-001952) is deposited in the herbarium of the Department of Pharmaceutical Sciences of Natural Resources, Kyoto Pharmaceutical University, Japan.Cells MK-1 cells were provided by Prof. M. Katano of Faculty of Medicine, Kyushu University, and HeLa and Determination of Antiproliferative Activity Inhibition of the cellular growth was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay described by Mosmann.12) The detailed procedure is shown in the previous paper. 13)Extraction and Isolation The dried roots (1.5 kg) of Inula helenium grown in Tibet were extracted with MeOH at room temperature for 2 weeks. The MeOH extract was then filtered and evaporated under reduced pressure to obtain a viscous mass (555.1 g). This material was suspended with H 2 O and partitioned with n-hexane, CHCl 3 , EtOAc and nBuOH to give each organic fraction, respectively. The antiproliferative activities (GI 50 mg/ml) of these organic fractions are shown in Fig. 1. The H 2 O fraction exhibited no activity.The n-hexane fraction (142 g) was subjected to column chromatography on silica gel (800 g) to give 7 fractions using an increasi...