Kiyo Shimada

CRELD2 is a novel endoplasmic reticulum stress-inducible gene.

Recently, endoplasmic reticulum (ER) stress responses have been suggested to play important roles in maintaining various cellular functions and to underlie many tissue dysfunctions. In this study, we first identified cysteine-rich with EGF-like domains 2 (CRELD2) as an ER stress-inducible gene by analyzing a microarray analysis of thapsigargin (Tg)-inducible genes in Neuro2a cells. CRELD2 mRNA is also shown to be immediately induced by treatment with the ER stress-inducing reagents tunicamycin and brefeldin A. In the genomic sequence of the mouse CRELD2 promoter, we found a typical ER stress responsible element (ERSE), which is well conserved among various species. Using a luciferase reporter analyses, we demonstrated that the ERSE in mouse CRELD2 is functional and responds to Tg and ATF6-overexpression. Each mutation of ATF6- or NF-Y-binding sites in the ERSE of the mouse CRELD2 promoter dramatically decreased both the basal activity and responsiveness toward the ER stress stimuli. Our study suggests that CRELD2 could be a novel mediator in regulating the onset and progression of various ER stress-associated diseases.

Repeated Methamphetamine Administration Alters Expression of the NMDA Receptor Channel ɛ2 Subunit and Kinesins in the Mouse Brain

Abstract:  Repeated amphetamine administration results in behavioral sensitization. Behavioral sensitization related to abuse and/or relapse may be associated with stable changes in gene expression. To explore the participating genes, we examined the changes in gene expression levels 24 h or 21 days (long‐term withdrawal period) after chronic methamphetamine (METH) treatment for 2 weeks. The expression of several genes related to glutamatergic neural transmission was altered, although changes in the corresponding protein expression were not always consistent with the results for mRNA expression. Of interest, in the frontal cortex of mice treated with METH for 2 weeks, protein expression levels of KIF17 and the N‐methyl‐D‐asparate (NMDA) receptor channel ɛ2 subunit (NRɛ2) were concomitantly increased. The alteration in expression of these proteins, KIF17 and NRɛ2, might be a part of the molecular basis of the behavioral sensitization to METH.

Changes in Expression of the Mouse Homologues of KIAA Genes after Subchronic Methamphetamine Treatment

Abstract: Amphetamine abuse may be associated with adaptive changes in gene expression in the brain. In the present study, a newly developed cDNA array system comprising mouse KIAA (mKIAA) cDNA clones was used to examine the gene expression affected by chronic methamphetamine treatment. Approximately 800 mKIAA clones were blotted onto a nylon membrane and hybridized with 33P‐labeled cDNA derived from mRNAs isolated from the whole brains of mice that had been treated daily with saline or methamphetamine (2 mg/kg, i.p.) for 2 weeks. The arrays displayed robust hybridization for almost all transcripts. The results obtained from five experiments were averaged, each performed with triplicate samples. Several clones were chosen as positive candidates for methamphetamine‐induced changes; however, only Per2 and mKIAA0099 genes showed a significantly increased expression (P < .05). Subsequently, with the focus on the period‐related proteins, the expression of these proteins in various parts of the rat brain were assessed by immunoblot analysis. Chronic administration of methamphetamine (8 mg/kg, i.p., for 10 days) caused increased Per2 protein expression in the hippocampus. Interestingly, chronic methamphetamine treatment at a lower dose (4 mg/kg, i.p., for 10 days) induced an increase in SCN circadian oscillatory protein (SCOP) expression, also in the hippocampus. These data suggest that long‐lasting alterations of the period‐related gene expressions in the hippocampus might play an important role in methamphetamine addiction.

Role of an ER stress response element in regulating the bidirectional promoter of the mouse CRELD2 - ALG12 gene pair

BackgroundRecently, we identified cysteine-rich withEGF-likedomains 2 (CRELD2) as a novel endoplasmic reticulum (ER) stress-inducible gene and characterized its transcriptional regulation by ATF6 under ER stress conditions. Interestingly, the CRELD2 and asparagine-linkedglycosylation 12 homolog (ALG12) genes are arranged as a bidirectional (head-to-head) gene pair and are separated by less than 400 bp. In this study, we characterized the transcriptional regulation of the mouse CRELD2 and ALG12 genes that is mediated by a common bidirectional promoter.ResultsThis short intergenic region contains an ER stress response element (ERSE) sequence and is well conserved among the human, rat and mouse genomes. Microarray analysis revealed that CRELD2 and ALG12 mRNAs were induced in Neuro2a cells by treatment with thapsigargin (Tg), an ER stress inducer, in a time-dependent manner. Other ER stress inducers, tunicamycin and brefeldin A, also increased the expression of these two mRNAs in Neuro2a cells. We then tested for the possible involvement of the ERSE motif and other regulatory sites of the intergenic region in the transcriptional regulation of the mouse CRELD2 and ALG12 genes by using variants of the bidirectional reporter construct. With regards to the promoter activities of the CRELD2-ALG12 gene pair, the entire intergenic region hardly responded to Tg, whereas the CRELD2 promoter constructs of the proximal region containing the ERSE motif showed a marked responsiveness to Tg. The same ERSE motif of ALG12 gene in the opposite direction was less responsive to Tg. The direction and the distance of this motif from each transcriptional start site, however, has no impact on the responsiveness of either gene to Tg treatment. Additionally, we found three putative sequences in the intergenic region that antagonize the ERSE-mediated transcriptional activation.ConclusionsThese results show that the mouse CRELD2 and ALG12 genes are arranged as a unique bidirectional gene pair and that they may be regulated by the combined interactions between ATF6 and multiple other transcriptional factors. Our studies provide new insights into the complex transcriptional regulation of bidirectional gene pairs under pathophysiological conditions.

High-Throughput Production of the Recombinant Proteins Expressed in Escherichia coli Utilizing cDNA Resources

Conventionally, expression plasmids in Escherichia coli have generally been constructed using ligation reaction-assisted cloning followed by the generation of inserts. In such cases, the insert was generated by polymerase chain reaction (PCR), digestion using restriction enzymes, or oligonucleotide synthesis. To overcome the restrictions of these conventional methods, we improved them by utilizing an in vitro site-specific recombination reaction, based on the integrase-excisionase system of bacteriophage lambda to insert DNA fragments. This method enabled us to insert tens of fragments into expression vectors in parallel. We applied these methods to produce glutathione S-transferase (GST)-fused or maltose-binding protein (MBP)-fused proteins in Escherichia coli. As a result, we successfully produced and purified more than 3,000 recombinant proteins for further study of reverse chemical genetics.