Increased vulnerability of hippocampal pyramidal neurons to the toxicity of kainic acid in OASIS-deficient mice

The endoplasmic reticulum (ER) stress response is a defense system for dealing with the accumulation of unfolded proteins in the ER lumen. Old astrocyte specifically induced substance (OASIS) is known to be expressed in astrocytes and involved in the ER stress response; however the function of OASIS in the injured brain has remained unclear. In this study, we examined the roles of OASIS in neuronal degeneration in the hippocampi of mice intraperitoneally injected with kainic acid (KA). OASIS mRNA was strongly induced in response to KA injection, with a similar time course to the induction of ER molecular chaperone immunoglobulin heavy chain binding protein mRNA. In situ hybridization showed that KA injection causes induction of immunoglobulin heavy chain binding protein mRNA in glial fibrillary acidic protein-positive astrocytes as well as in pyramidal neurons, although up-regulation of OASIS mRNA was only detected in glial fibrillary acidic protein-positive astrocytes. Primary cultured astrocytes, but not the neurons of OASIS −/− mice, revealed reduced vulnerability to ER stress. Furthermore, pyramidal neurons in the hippocampi of OASIS −/− mice were more susceptible to the toxicity induced by KA than those of wild-type mice. Taken together, these data suggest that OASIS expressed in astrocytes plays important roles in protection against the neuronal damage induced by KA.     

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AimsSeveral recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.Main methodsThe gastric mucosa was removed from Sprague–Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.Key findingsSpecific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.SignificanceDuring the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.     

Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants

Metabolomics is an ‘omics’ approach that aims toanalyze all metabolites in a biological sample comprehensively.The detailed metabolite profiling of thousands of plant sampleshas great potential for directly elucidating plant metabolicprocesses. However, both a comprehensive analysis and a highthroughput are difficult to achieve at the same time due tothe wide diversity of metabolites in plants. Here, we have establisheda novel and practical metabolomics methodology for quantifyinghundreds of targeted metabolites in a high-throughput manner.Multiple reaction monitoring (MRM) using tandem quadrupole massspectrometry (TQMS), which monitors both the specific precursorions and product ions of each metabolite, is a standard techniquein targeted metabolomics, as it enables high sensitivity, reproducibilityand a broad dynamic range. In this study, we optimized the MRMconditions for specific compounds by performing automated flowinjection analyses with TQMS. Based on a total of 61,920 spectrafor 860 authentic compounds, the MRM conditions of 497 compoundswere successfully optimized. These were applied to high-throughputautomated analysis of biological samples using TQMS coupledwith ultra performance liquid chromatography (UPLC). By thisanalysis, approximately 100 metabolites were quantified in eachof 14 plant accessions from Brassicaceae, Gramineae and Fabaceae.A hierarchical cluster analysis based on the metabolite accumulationpatterns clearly showed differences among the plant families,and family-specific metabolites could be predicted using a batch-learningself-organizing map analysis. Thus, the automated widely targetedmetabolomics approach established here should pave the way forlarge-scale metabolite profiling and comparative metabolomics.     

Requirement of the SH4 and tyrosine-kinase domains but not the kinase activity of Lyn for its biosynthetic targeting to caveolin-positive Golgi membranes

Background

The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated.

Methods

Subcellular localization of Lyn and its mutants was determined by confocal microscopy.

Results

We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes.

General significance

Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity.     

Structure–activity relationship study on polyglutamine binding peptide QBP1

Aggregation and deposition of expanded polyglutamine proteins in the brain cause neurodegenerative diseases including Huntington disease. This pathogenic process is suppressed and delayed in the presence of polyglutamine binding peptide 1 (QBP1), which we previously identified as an undecapeptide binding to pathogenic polyglutamine proteins from phage display peptide libraries. In this paper, a structure–activity relationship study on QBP1 was conducted to determine the pharmacophores for inhibition of polyglutamine aggregation. Furthermore, a truncation study identified an octapeptide as the minimum structure for suppressing aggregation of polyglutamine proteins, which is equipotent to the parent undecapeptide QBP1.     

Development of novel cell surface display in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using porin

We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used α-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.     

Discovery of selective PDE4B inhibitors

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.