Biomedical subjects
Publications and source records attributed to M Mishina.
The primary structure of the mouse glutamate receptor beta 2 subunit has been deduced by cloning and sequencing cDNA. The beta 2 subunit has structural characteristics common to the subunits of glutamate-gated ion channels. Expression of the cloned cDNA in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.
The presence and primary structure of a novel subunit of the mouse glutamate receptor channel, designated as gamma 2, have been revealed by cloning and sequencing the cDNA. The gamma 2 subunit has structural characteristics common to the neurotransmitter-gated ion channel family and shares a high amino acid sequence identity with the rat KA-1 subunit, thus constituting the gamma subfamily of the glutamate receptor channel. Expression of the gamma 2 subunit together with the beta 2 subunit in Xenopus oocytes yields functional glutamate receptor channels selective for kainate.
In situ hybridization analyses have revealed drastic changes in expression and distribution of five subunit mRNAs of the mouse NMDA receptor channel during brain development. The epsilon 1 subunit mRNA is expressed postnatally and widely in the brain. On the other hand, the epsilon 2 subunit mRNA is found throughout the entire embryonic brain, but its expression becomes restricted to the forebrain at postnatal stages. The epsilon 3 subunit mRNA appears postnatally and predominantly in the cerebellum, whereas the epsilon 4 subunit mRNA is abundantly expressed in the diencephalon and the brainstem at embryonic and neonatal stages. In contrast, the zeta 1 subunit mRNA distributes ubiquitously in the brain throughout development. These findings suggest that changes in the subunit composition of the NMDA receptor channel take place during brain development.
Using a baculovirus expression vector system, the alpha 1 subunit of the mouse AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-selective glutamate receptor channel was expressed in insect Spdoptera frugiperda cells. Binding studies using [3H]AMPA showed that insect cells infected with the recombinant virus expressed approximately 1.8 x 10(5) binding sites per cell on their surface. The ligand binding characteristics of the receptors expressed in insect cells were examined. The baculovirus-insect cell expression system affords high-efficiency expression of the receptor in sufficient amounts to permit structural and functional analyses.
Functional analysis of AMPA-selective glutamate receptor channels expressed in Xenopus oocytes from cloned cDNAs has shown that homomeric channels formed by the GluR1 subunit are permeable to Ca2+, whereas heteromeric channels composed of the GluR1 and GluR2 subunits show little permeability. Furthermore, substitution of glutamine for arginine in putative transmembrane segment M2 of the GluR2 subunit makes the heteromeric channels permeable to Ca2+. These results suggest that the GluR2 subunit plays a key role in keeping AMPA-selective glutamate receptor channels essentially impermeable to Ca2+ and that the critical determinant is the positively charged residue in M2 segment.
The channel pore of the nicotinic acetylcholine receptor (AChR) has been investigated by analysing single-channel conductances of systematically mutated Torpedo receptors expressed in Xenopus oocytes. The mutations mainly alter the size and polarity of uncharged polar amino acid residues of the acetylcholine receptor subunits positioned between the cytoplasmic ring and the extracellular ring. From the results obtained, we conclude that a ring of uncharged polar residues comprising threonine 244 of the alpha-subunit (alpha T244), beta S250, gamma T253 and delta S258 (referred to as the central ring) and the anionic intermediate ring, which are adjacent to each other in the assumed alpha-helical configuration of the M2-containing transmembrane segment, together form a narrow channel constriction of short length, located close to the cytoplasmic side of the membrane. Our results also suggest that individual subunits, particularly the gamma-subunit, are asymmetrically positioned at the channel constriction.
In some patients with paraneoplastic cerebellar degeneration (PCD), autoantibodies against neural components have been identified. Here, we demonstrate a major 58 kd protein antigen in an immunoblot of human cerebellum by serum from a patient with PCD. Immunohistochemically, the serum recognized neural cells especially Purkinje cells in a human brain. To identify the details of the target antigens for the antibody, we isolated a cDNA clone from a human cerebellar library. Homology searches revealed a similarity with the zinc finger proteins. PCD related proteins reported here may be important to maintain neural cells especially those in the cerebellum, and further studies on this molecule may help us elucidate the causes of degenerative or autoimmune diseases in the cerebellum.
The mouse myelin-associated glycoprotein gene was isolated from a mouse gene library. This gene was split into 13 exons distributed about 15 kb in length. Each extracellular immunoglobulin-related domain was encoded by a single exon, and RNA splicing between those exons occurred between the first and second nucleotides of the junctional codon, the features of which are conserved in most of the genes of the immunoglobulin superfamily. The sequence of the 5'-flanking region appeared to have some regions homologous to other myelin proteins, which suggested that they were possible cis-elements for specific expression of oligodendrocytes.
To gain an insight into the molecular basis of the weak but significant selectivity among alkali metal cations of the nicotinic acetylcholine receptor (AChR) channel, we have determined single-channel conductance and permeability ratios for alkali metal cations on specifically mutated Torpedo californica AChR channels expressed in Xenopus oocytes. The mutations involved charged and polar side chains in the three anionic rings (extracellular, intermediate and cytoplasmic ring) which have previously been found to determine the rate of K+ transport through the AChR channel. The results obtained reveal that mutations in the intermediate ring exert much stronger effects on ion selectivity than do mutations in the extracellular and the cytoplasmic ring. The experimental results, together with simulations of the channel's energy profile, suggest that the amino acid residues forming the intermediate ring come into close contact with permeating cations and possibly represent part of the physical correlate of the postulated selectivity filter in the AChR channel.
We report a case of huge renal arteriovenous aneurysm, which was successfully treated with transcatheter arterial embolization (TAE). A 72-year-old woman was admitted with the clinical symptom of dyspnea on effort. She felt a tremor in the left abdomen. Computerized tomographic scan showed a large enhanced mass (8x7x7 cm) on the left kidney. Selective renal arteriography revealed a renal arteriovenous aneurysm. Transcatheter arterial embolization with a steel coil was performed with minimal loss of renal parenchyma. A total of 270 cases of arteriovenous fistula have been reported in Japan and were statistically analyzed. The number of reports on TAE has dramatically increased since 1978, TAE can be first indicated for treatment of renal arteriovenous fistula in view of less invasiveness and preservation of renal function.
The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.
During the development of mammalian muscle the gamma-subunit of the nicotinic acetylcholine receptor (AChR) is replaced by the epsilon-subunit to produce well-defined alterations in the conductance and gating of the channel. To gain a better understanding of the functional role of the gamma- and epsilon-subunits, we have studied the properties of an AChR channel lacking these subunits. The AChR expressed in Xenopus oocytes injected with the bovine alpha-, beta- and delta-subunit-specific mRNAs (referred to as alpha beta delta-AChR) is unusual in that its channel opens spontaneously at a high frequency in the absence of agonist. From a comparison of the alpha beta delta-AChR with complete receptors containing either the gamma- or epsilon-subunit, we conclude that the gamma- and epsilon-subunits influence most channel properties, including agonist binding, and are especially important for stabilizing the closed state of the unliganded receptor channel. The alpha beta delta-AChR can form when a complete set of four subunit-specific mRNAs is injected. The ease with which it is assembled raises the possibility that the alpha beta delta-AChR contributes to some of the variations in receptor properties that occur during development.
A new selective and differential agar medium, polymyxin-mannose-tellurite (PMT) agar was devised to differentiate easily colonies of Vibrio cholerae O1 from those of V. cholerae non-O1. The differentiation between colonies of the two vibrios is based on mannose-fermentation. Colonies of V. cholerae O1 on the agar are agglutinated with O1 antiserum of V. cholerae much more easily than those on thiosulfate-citrate-bile salts-sucrose (TCBS) agar.
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Five odontogenic keratocysts were examined by using a scanning electron microscope (SEM) on the three-dimensional ultrastructure of epithelial lining cells. The fractured surface of the specimens showed a single row of columnar or cuboidal cells perpendicular to the flat basal lamina, a four- to five-cell layer of spinous cells, and a superficial layer of flattened parakeratinized cells. At high magnification of superficial cell surface, microvillis, short discontinuous branched or anastomosing microridges were observed.
Attempts have been made by means of recombinant DNA technology to understand the molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor (mAChR). Molecularly defined mAChR subtypes have been produced from the cloned DNAs in Xenopus oocytes and NG108-15 neuroblastoma-glioma hybrid cells as transient and stable expression systems, respectively, and agonist-induced cellular responses have been examined. The results obtained provide evidence that mAChR subtypes are selectively coupled with different effector systems, albeit not exclusively.
Chimaeric muscarinic acetylcholine receptors (mAChR) in which corresponding portions of mAChR I and mAChR II are replaced with each other have been produced in Xenopus oocytes by expression of cDNA constructs encoding them. Functional analysis of the chimaeric mAChRs indicates that a region mostly comprising the putative cytoplasmic portion between the proposed transmembrane segments V and VI is involved in selective coupling of mAChR I and mAChR II with different effector systems. In contrast, the exchange of this region between mAChR I and mAChR II does not significantly affect the antagonist binding properties of the two mAChR subtypes.
Muscarinic acetylcholine receptor (mAChR) III expressed in Xenopus oocytes, like mAChR I, mediates activation of a Ca2+-dependent Cl- current, whereas mAChR IV, like mAChR II, principally induces activation of Na+ and K+ currents in a Ca2+-independent manner. mAChR III has a sensitivity to agonist of about one order of magnitude higher than that of mAChR I in mediating the Ca2+-dependent current response in Xenopus oocytes and in stimulating phosphoinositide hydrolysis in NG108-15 neuroblastoma-glioma hybrid cells. The agonist-binding affinity of mAChR III is also about one order of magnitude higher than that of mAChR I.