Junko Iida

Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.

Lung alveolar epithelial cells are comprised of type I (ATI) and type II (ATII) cells. AΤΙ cells are polarized, although they have very flat morphology. The identification of marker proteins for apical and basolateral membranes of ATI cells is important to investigate into the differentiation of ATI cells. In this paper, we characterized receptor for advanced glycation end‐products (RAGE) as a marker for ATI cells. RAGE was localized on basolateral membranes of ATI cells in the immunoelectron microscopy and its expression was enhanced in a parallel manner to the differentiation of ATI cells in vivo and in primary cultures of ATII cells. RAGE and T1α, a well‐known ATI marker protein, were targeted to basolateral and apical membranes, respectively, when expressed in polarized Madine Darby canine kidney cells. Moreover, RAGE was expressed in ATI cells after T1αin vivo and in ex in vivo organ cultures. In conclusion, RAGE is a marker for basolateral membranes of well‐differentiated ATI cells. ATI cells require some signal provided by the in vivo environment to express RAGE.

Synaptic scaffolding molecule is involved in the synaptic clustering of neuroligin.

S-SCAM has a similar molecular organization to PSD-95. Both of them interact with a cell adhesion molecule, neuroligin. We previously reported that beta-catenin binds S-SCAM and recruits it to synapses. We have here examined using rat primary cultured neurons whether neuroligin recruits S-SCAM to synapses or S-SCAM determines the localization of neuroligin. Overexpressed neuroligin formed larger clusters under co-expression of S-SCAM but not of PSD-95. Overexpressed neuroligin blocked synaptic accumulation of PSD-95 but not of S-SCAM. S-SCAM mutant containing the neuroligin-binding region interfered with synaptic accumulation of neuroligin and PSD-95, whereas the similar mutant of PSD-95 had no effect. Biochemical studies revealed that neuroligin forms a ternary complex with S-SCAM and PSD-95 through manifold interactions. These findings imply that S-SCAM is tethered by beta-catenin to synapses and induces synaptic accumulation of neuroligin, which subsequently recruits PSD-95 to synapses.

Synaptic scaffolding molecule (S-SCAM) membrane-associated guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons

Synaptic scaffolding molecule (S‐SCAM) is a synaptic protein, which harbors five or six PSD‐95/Discs large/ZO‐1 (PDZ), a guanylate kinase and two WW domains. It interacts with NMDA receptor subunits, neuroligin and β‐catenin, and is involved in the accumulation of neuroligin at excitatory synapses. In this study, we have demonstrated S‐SCAM is localized at inhibitory synapses in rat primary cultured hippocampal neurons. We have identified β‐dystroglycan (β‐DG) as a binding partner for S‐SCAM at inhibitory synapses. WW domains of S‐SCAM bind to three sequences of β‐DG. We have also revealed that S‐SCAM can interact with neuroligin 2, which is known to be exclusively localized at inhibitory synapses. The WW domains and the second PDZ domain of S‐SCAM are involved in the interaction with neuroligin 2. β‐DG, neuroligin 2 and S‐SCAM form a tripartite complex in vitro. Neuroligin 2 is detected in the immunoprecipitates by anti‐β‐DG antibody from rat brain. S‐SCAM, β‐DG and neuroligin 2 are partially co‐localized in rat hippocampal neurons. These data suggest that S‐SCAM is associated with β‐DG and neuroligin 2 at inhibitory synapses, and functions as a linker between the dystrophin glycoprotein complex and the neurexin–neuroligin complex.

Synaptic Scaffolding Molecule α Is a Scaffold To Mediate N-Methyl-d-Aspartate Receptor-Dependent RhoA Activation in Dendrites

ABSTRACT Synaptic scaffolding molecule (S-SCAM) interacts with a wide variety of molecules at excitatory and inhibitory synapses. It comprises three alternative splicing variants, S-SCAMα, -β, and -γ. We generated mutant mice lacking specifically S-SCAMα. S-SCAMα-deficient mice breathe and feed normally but die within 24 h after birth. Primary cultured hippocampal neurons from mutant mice have abnormally elongated dendritic spines. Exogenously expressed S-SCAMα corrects this abnormal morphology, while S-SCAMβ and -γ have no effect. Active RhoA decreases in cortical neurons from mutant mice. Constitutively active RhoA and ROCKII shift the length of dendritic spines toward the normal level, whereas ROCK inhibitor (Y27632) blocks the effect by S-SCAMα. S-SCAMα fails to correct the abnormal spine morphology under the treatment of N-methyl-d-aspartate (NMDA) receptor inhibitor (AP-5), Ca2+/calmodulin kinase inhibitor (KN-62), or tyrosine kinase inhibitor (PP2). NMDA treatment increases active RhoA in dendrites in wild-type hippocampal neurons, but not in mutant neurons. The ectopic expression of S-SCAMα, but not -β, recovers the NMDA-responsive accumulation of active RhoA in dendrites. Phosphorylation of extracellular signal-regulated kinase 1/2 and Akt and calcium influx in response to NMDA are not impaired in mutant neurons. These data indicate that S-SCAMα is a scaffold required to activate RhoA protein in response to NMDA receptor signaling in dendrites.

Profiling and biomarker identification in plasma from different Zucker rat strains via high mass accuracy multistage mass spectrometric analysis using liquid chromatography/mass spectrometry with a quadrupole ion trap-time of flight mass spectrometer.

High mass accuracy electrospray ionisation multistage tandem mass spectrometry (MS(n)) was applied to metabolite profiling studies on plasma samples derived from two strains of rat (the Zucker (fa/fa) obese strain and the normal wild type). Using a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer, metabolite profiling software was applied to locate components of biological significance that could account for the differences between the two strains of rat and a formula prediction software tool was used to help identify individual components. The primary factor discriminating between the two populations was the concentration of endogenous lipids. In the Zucker (fa/fa) obese strain, the dominant ion signals and MS(n) spectra were in agreement with lysoglycerophosphocholine components such as palmitoyllysophosphatidylcholine, 1-oleoylglycerophosphocholine, 1-octadecyl-sn-glycero-3-phosphocholine and 1-stearoylglycerophosphocholine and these were found in relatively higher concentrations compared to the normal wild type. Components were identified using high mass accuracy MS(n) data, formula prediction software and by agreement with published mass spectra through internet databases, rather than using a conventional approach with authentic standards. This application shows that the use of high mass accuracy electrospray ionisation MS(n) together with a software tool can be used effectively to detect and characterise unknown analytes in complex matrices, and represents a promising approach for future profiling studies.

Synaptic scaffolding molecule interacts with axin.

Synaptic scaffolding molecule (S‐SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N‐methyl‐d‐aspartate receptor subunits, neuroligin, and β‐catenin. Here, we identified Axin as a novel binding partner of S‐SCAM. Axin was co‐immunoprecipitated with S‐SCAM from rat brain, detected in the post‐synaptic density fraction in rat brain subcellular fractionation, and partially co‐localized with S‐SCAM in neurons. The guanylate kinase domain of S‐SCAM directly bound to the GSK3β‐binding region of Axin. S‐SCAM formed a complex with β‐catenin and Axin, but competed with GSK3β for Axin‐binding. Thereby, S‐SCAM inhibited the Axin‐mediated phosphorylation of β‐catenin by GSK3β.

Synaptic localization of SAPAP1, a synaptic membrane-associated protein.

Background: SAPAP1 was originally identified as a protein interacting with the guanylate kinase domain of PSD‐95. SAPAP1 also interacts with various proteins, including neurofilaments, synaptic scaffolding molecule (S‐SCAM), nArgBP2, dynein light chain and Shank through different regions.

Identification of a new minor iridoid glycoside in Symplocos glauca by thermospray liquid chromatography-mass spectrometry.

Abstract Iridoid glycosides in Symplocos glauca were identified by thermospray liquid chromatography-mass spectrometry. The conditions for optimization of the separation of iridoid glycosides were investigated. The mass spectra obtained gave qualitative information concerning both the aglycone and sugar moieties and also the molecular weights. Methanol extracts from the leaves of S. glauca were analysed for iridoid glycosides. Not only major but also minor components could be separated and identified from the mass spectra and mass chromatograms. A new minor component in S. glauca was tentatively identified as 6-dihydroverbenalin.

Synaptic localization of membrane‐associated guanylate kinase‐interacting protein mediated by the pleckstrin homology domain

Membrane‐associated guanylate kinase‐interacting protein (MAGUIN) has been identified as a protein binding postsynaptic density (PSD)‐95 and synaptic scaffolding molecule (S‐SCAM). MAGUIN has one sterile α motif, one conserved region in connector enhancer of ksr (Cnk) (CRIC), one PSD‐95/Dlg‐A/ZO‐1 (PDZ) and one pleckstrin homology (PH) domain. There are two isoforms, MAGUIN‐1 and ‐2. MAGUIN‐1 binds the PDZ domains of PSD‐95 and S‐SCAM by the C‐terminus, whereas MAGUIN‐2 does not bind to PSD‐95 or S‐SCAM. Here, we have determined that MAGUIN‐2 is also localized at synapses and that the synaptic localization of MAGUIN depends on the pleckstrin homology domain. The overexpressed C‐terminal PDZ‐binding region inhibits the synaptic targeting of PSD‐95. Furthermore, the synaptic targeting of MAGUIN does not require N‐methyl‐d‐aspartate (NMDA) receptor activity. These findings suggest that MAGUIN‐1 and ‐2 are recruited to synapses by the PH domain and that MAGUIN‐1 subsequently interacts with PSD‐95 at synapses.

1-Cyanoisoindole derivative as a highly sensitive fluorescence labelling reagent for steroidal primary alcohols in liquid chromatography

Abstract A sensitive and reactive labelling reagent for steroidal primary alcohols, m -(1-cyano-2-isoindole)benzoyl azide, was prepared from the corresponding carboxylic acid, which was synthesized from o -phthalaldehyde and m -aminobenzoic acid in the presence of potassium cyanide in one step. A hydroxysteroid, such as cortisol, can react with the reagent, when kept in benzene at 80 °C for 40 min, resulting in the formation of a urethane derivative which shows a single peak on the chromatogram and provides high sensitivity. The detection limit of the cortisol in reversed-phase chromatography is 80 fmol.