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Inflammation has been associated with depression, and innate immune receptors, such as the Toll-like receptor (TLR) 2/4 in the medial prefrontal cortex (mPFC), are crucial for chronic stress-induced depression-related behaviors in mice. HMGB1, a putative ligand for TLR2/4, has been suggested to promote depression-related behaviors under acute stress. However, the roles of endogenous HMGB1 under chronic stress remain to be investigated. Here, we found that the cerebroventricular infusion of HMGB1 proteins blocked stress-induced social avoidance and that HMGB1-neutralizing antibodies augmented repeated social defeat stress-induced social avoidance in mice, suggesting the antidepressive-like effect of HMGB1 in the brain. By contrast, the infusion of HMGB1-neutralizing antibodies to the mPFC and HMGB1 knockout in α-CaMKII-positive forebrain neurons attenuated the social avoidance, suggesting the pro-depressive-like effect of HMGB1 released from prefrontal neurons under chronic stress. In addition, repeated social defeat stress induced HMGB1 nuclear export selectively in mPFC neurons, which was abolished in the mice lacking RAGE, one of HMGB1 receptors, suggesting the positive feedback loop of HMGB1-RAGE signaling under chronic stress. These findings pave the way for identifying multiple roles of HMGB1 in the brain for chronic stress and depression.

Prostaglandin E2 (PGE2 ) plays crucial roles in managing microglial activation through the prostanoid EP2 receptor, a PGE2 receptor subtype. In this study, we report that PGE2 enhances interferon-γ (IFN-γ)-induced nitric oxide production in microglia. IFN-γ increased the release of nitrite, a metabolite of nitric oxide, which was augmented by PGE2 , although PGE2 by itself slightly affects nitrite release. The potentiating effect of PGE2 was positively associated with increased expression of inducible nitric oxide synthase. In contrast to nitrite release induced by IFN-γ, lipopolysaccharide-induced nitrite release was not affected by PGE2 . An EP2 agonist, ONO-AE1-259-01 also augmented IFN-γ-induced nitrite release, while an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, or an EP4 agonist, ONO-AE1-329, did not. In addition, the potentiating effect of PGE2 was inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3-240, or an EP4 antagonist, ONO-AE3-208, at 10-6 M. Among the EP agonists, ONO-AE1-259-01 alone was able to accumulate cyclic adenosine monophosphate (AMP), and among the EP antagonists, PF-04418948 was the only one able to inhibit PGE2 -increased intracellular cyclic AMP accumulation. On the other hand, IFN-γ promoted phosphorylation of signal transducer and activator of transcription 1, which was not affected by PGE2 . Furthermore, other prostanoid receptor agonists, PGD2 , PGF2α , iloprost, and U-46119, slightly affected IFN-γ-induced nitrite release. These results indicate that PGE2 potentiates IFN-γ-induced nitric oxide production in microglia through the EP2 receptor, which may shed light on one of the pro-inflammatory aspects of PGE2 .

Background: Cyclin G1 is a transcriptional target of p53 and is induced by DNA damage in a p53 dependent manner. Analysis of cyclin G1 disrupted mice demonstrated that cyclin G1 is involved in many of the functions regulated by p53 such as apoptosis, growth control and check point regulation in response to DNA damage. The results suggest that the main role of cyclin G1 is to mediate or regulate the function of p53. Results: Western blot analysis revealed that the accumulation of p53 protein during the initial 24 h period following DNA damage is reduced in cyclin G1–/– cells compared to wild-type cells. This decrease in p53 accumulation could be recovered by introducing a cDNA expressing cyclin G1. Cyclin G1 interacted directly with MDM2 and promoted the formation of the ARF/MDM2 complex within the initial 24 h period following DNA damage. Furthermore, 48 h after irradiation, accumulation of p53 protein was enhanced in cyclin G1–/– cells compared to wild-type cells. In contrast, in 48 h postirradiated wild-type cells, the cyclin G1-MDM2 complex was found not to be associated with ARF but with the B′α subunit of protein phosphatase A. Conclusion: These results suggest that cyclin G1 stabilizes and promotes the degradation of p53 protein by associating, respectively, with MDM2 complexes containing ARF and PP2A.

Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- MEFs in G2/M phase after irradiation was reduced by 50% relative to cyclin G1+/+ MEFs. Our results demonstrate that cyclin G1 plays roles in G2/M arrest, damage recovery and growth promotion after cellular stress.

We previously cloned a rat cDNA encoding GAK, an association partner of cyclin G and CDK5. Here, we report the cloning of a cDNA encoding human GAK (1311 amino acids) and show that all of the unique motifs that characterize rat GAK, such as the presence of a Ser/Thr kinase domain, a tensin/auxilin homologous domain, and a Tyr phosphorylation target site, are conserved. The expression profiles of GAK and cyclin G during the synchronized HeLa cell cycle showed that GAK expression oscillates slightly, peaking at G1 phase, although the histone H1 kinase activity remains constant throughout the cell cycle. We also found that the kinase activity of immunoprecipitates of anti-cyclin G antibody fluctuates during the cell cycle with a peak at G1 phase, although the expression level of cyclin G remains almost constant. Northern blot analysis showed that GAK is expressed ubiquitously, with the highest level of expression being observed in the testis. By the FISH technique, we assigned the chromosomal localization of GAK to 4p16.

We have cloned a cDNA encoding a novel association partner of cyclin G by West-Western blotting. The cDNA encodes a protein that harbors a Ser/Thr protein kinase-like catalytic domain at the N-terminal. Hence, we named it GAK (cyclin - ssociated inase). The long C-terminal extension shares homology with tensin and auxilin, and contains a leucine zipper region. Co-immunoprecipitation and Western blotting showed that GAK and cyclin G associate together in vivo. GAK also co-precipitated with CDK5, and CDK5 was found to be associated with cyclin G. We also showed by BIAcore analysis that the GAK-cyclin G interaction was direct.

The 2nd International Symposium on Earth History of Asia 31 October – 3 November, Niigata, Japan

RECS1 is a novel shear stress-responsive gene that encodes a protein putatively forming seven-span transmembrane domains. We reports here that mouse RECS1 (mRECS1) transcripts is detected in most tissues except for thymus, spleen and testis. The putative cytoplasmic N-terminus of mRECS1 has a high content of proline (23%) and glycine (12%) residues, contains one PPXY motif, multiple PXXP motifs and one overlapping P(T/S)AP and PPXY motif (P(T/S)APPXY). The PPXY motif lies within one potential PEST sequence (PEST score: +7.65). We prepared anti-RECS1 polyclonal antibody and found by western blot analysis that the mRECS1 protein in the lung and aorta was detected as a 34.4 kDa band. However, one shifted 58 kDa band or three shifted bands (48, 69, 82 kDa) were detected in the heart or the liver, respectively. Since northern blot detected only one species of mRECS1 mRNA in heart and liver tissues, as well as other tissues (approximately 2.2 kb), these differences in molecular weight seem to be due to posttranslational modification. Biochemical fractionation and RECS1-GFP fusion protein revealed that RECS1 localizes at the endosomal/lysosomal membranes in the cytoplasm. To understand the function of RECS1 in the body, we made RECS1 knockout (KO) mice and found that RECS1 KO mice (older than 14 months) are prone to cystic medial degeneration (CMD). Taken together, we conclude that RECS1 is an endosomal/lysosomal membrane protein which plays protective roles in vascular remodeling.

Cyclin G1 is a transcriptional target of p53 and is induced by DNA damage in a p53 dependent manner. Analysis of cyclin G1 disrupted mice demonstrated that cyclin G1 is involved in many of the functions regulated by p53 such as apoptosis, growth control and check point regulation in response to DNA damage. The results suggest that the main role of cyclin G1 is to mediate or regulate the function of p53.Western blot analysis revealed that the accumulation of p53 protein during the initial 24 h period following DNA damage is reduced in cyclin G1-/- cells compared to wild-type cells. This decrease in p53 accumulation could be recovered by introducing a cDNA expressing cyclin G1. Cyclin G1 interacted directly with MDM2 and promoted the formation of the ARF/MDM2 complex within the initial 24 h period following DNA damage. Furthermore, 48 h after irradiation, accumulation of p53 protein was enhanced in cyclin G1-/- cells compared to wild-type cells. In contrast, in 48 h postirradiated wild-type cells, the cyclin G1-MDM2 complex was found not to be associated with ARF but with the B'alpha subunit of protein phosphatase A.These results suggest that cyclin G1 stabilizes and promotes the degradation of p53 protein by associating, respectively, with MDM2 complexes containing ARF and PP2A.

Mouse genomic DNA harboring the full coding sequence of cyclin G1 was cloned and analyzed. The locations of five coding exons and the intron-exon boundary sequences were found to be conserved between the mouse and the human genes. Two putative binding sites for the p53 tumor suppressor gene product were found around the first exon: one was located in the 5' regulatory region, and the other was in the first intron. The mouse cyclin G1 gene was mapped to bands A5 to B1 of chromosomes 11 (11A5-B1) by FISH using genomic DNA clone as a biotinylated probe. The location of mouse cyclin G1 is syntenic to that of its human homologue, which we previously mapped to 5q32-q34 of chromosome 5. An additional faint signal was detected on chromosome 4 (4B1-C2), probably indicating the presence of a cyclin G1-related gene or pseudogene in the mouse genome.

We have cloned a cDNA encoding a novel association partner of cyclin G by West-Western blotting. The cDNA encodes a protein that harbors a Ser/Thr protein kinase-like catalytic domain at the N-terminal. Hence, we named it GAK (cyclin G - a ssociated k inase). The long C-terminal extension shares homology with tensin and auxilin, and contains a leucine zipper region. Co-immunoprecipitation and Western blotting showed that GAK and cyclin G associate together in vivo. GAK also co-precipitated with CDK5, and CDK5 was found to be associated with cyclin G. We also showed by BIAcore analysis that the GAK-cyclin G interaction was direct.

The concept of a polyp-cancer sequence is assuming increasing credibility as a factor in the development of colorectal cancer. Colonoscopy permits most colonic polyps to be endoscopically removed and studied pathologically. Of various polyp types encountered in the colon only neoplastic polyps are regarded as having malignant potential. Neoplastic polyps include tubular adenomas (formerly, adenomatous polyps), villous adenomas and villotubular adenomas (formerly, mixed or tuboglandular polyps). Cancerous changes must penetrate the muscularis mucosae for a polyp to be regarded as clinically malignant. The present report analyzes a series of 5,786 adenomas from over 7,000 polyp endoscopically removed. The largest number of each type of adenoma presented in the sigmoid colon, followed by the descending colon in terms of frequency. In all zones tubular adenomas were most common, villous least. Abnormal cellular change, from dysplasia to carcinoma in situ to invasive cancer was most frequently found in the sigmoid colon and, in all colon sectors, increased as the villous componency of the polyp increased. However, all categories of neoplastic polyps showed malignant changes. Polyp size, long recognized as a factor, was shown to be importantly related to malignant change, but invasive cancer was found even in polyps less than 1 cm in diameter. In addition, the incidence of malignancy rose parallel to the frequency of synchronous and metachronous polyps. A vigorous program for detection and endoscopic removal of colorectal polyps is recommended as a means of reducing the incidence of colorectal cancer.