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Purpose: Abnormal fundus autofluorescence (FAF) potentially precedes onset of late age-related macular degeneration (AMD) in Caucasian patients. Many differences exist between Asian and Caucasian patients regarding AMD types and severity, gender, and genetic backgrounds. We investigated the characteristics of abnormal FAF and retinal sensitivity in the fellow eyes of Japanese patients with unilateral neovascular AMD. Methods: Sixty-six patients with unilateral neovascular AMD and abnormal FAF in the fellow eye were enrolled in this multicenter, prospective, observational study. The best-corrected visual acuity, fundus photographs, FAF images, and retinal sensitivity on microperimetry were measured periodically for 12 months. The FAF images were classified into eight patterns based on the International Fundus Autofluorescence Classification Group. The points measured by microperimetry were superimposed onto the FAF images and fundus photographs and classified as “within,” “close,” and “distant,” based on the distance from the abnormal FAF and other findings. The relationship between the location of the baseline abnormal FAF and retinal sensitivity was investigated. Results: In Japanese patients, patchy (33.3%) and focally increased (30.3%) patterns predominated in the abnormal FAF. Intermediate-to-large drusen was associated predominantly with hyperfluorescence and hypofluorescence. Neovascular AMD developed within 1 year in six (9.1%) eyes, the mean baseline retinal sensitivity of which was 12.8 ± 4.7 dB, significantly (p<0.002) lower than the other eyes. In 44 of the other 60 eyes, microperimetry was measurable at baseline and month 12 and the mean retinal sensitivity improved significantly from 13.5 ± 4.4 to 13.9 ± 4.8 dB (p<0.001), possibly associated with lifestyle changes (e.g., smoking cessation, antioxidant and zinc supplementation). The mean retinal sensitivities of points within and close to the abnormal FAF were 9.9 and 11.7 dB, respectively, which were significantly lower than the 14.0 dB of the points distant from the abnormal FAF. Conclusion: In Japanese patients, patchy and focally increased patterns predominated in the abnormal FAF. The retinal sensitivity was lower close to/within the abnormal FAF. FAF and microperimetry are useful to assess macular function before development of neovascular AMD or geographic atrophy. [ABSTRACT FROM AUTHOR]

Among various tyrosine kinases, a family of Janus kinases (JAK) has been elucidated as key players in signal transduction from vital cytokine receptors, such as interleukins and interferons. Indeed, recent rapid progress in JAK inhibitors in addition to biological agents provided therapeutic options for various diseases, including immune-mediated inflammatory diseases and hematological disorders. Efforts have culminated in the approval of nine JAK inhibitors in Japan in the recent decade. The safety profiles of JAK inhibitors seem to largely depend on patient populations and drug dosing, rather than on JAK selectivity. Thus, the comparison of various disease indications is pivotal for the understanding and proper use of JAK inhibitors.

I would like to congratulate the Journal of Biochemistry (JB) on its 100th anniversary. I served as the Editor-in-Chief for four years starting in 2010 and succeeded Dr Naoyuki Taniguchi. [ABSTRACT FROM AUTHOR]

Purpose: Genomic profiling programs have been implemented to apply next-generation sequencing (NGS) for facilitating trial enrollment. SCRUM-Japan GI-SCREEN is a large-scale genomic profiling program in advanced gastrointestinal cancers using a validated genomic assay with the goal of facilitating enrollment in targeted clinical trials, generating real-world data, and performing clinicogenomic analysis for biomarker discovery., Patients and Methods: Genotyping of tumor tissue samples from 5,743 patients with advanced gastrointestinal cancers enrolled in GI-SCREEN was centrally performed with NGS. Patients were enrolled in matched trials of targeted agents affiliated with GI-SCREEN on the basis of genotyping results., Results: A total of 11 gastrointestinal cancers were included, with colorectal cancer being the most common. The median age ranged from 59 to 70.5 years across cancer types. Patients enrolled after initiation of first-line treatment had significantly longer overall survival (OS) than that before treatment initiation with a median survival time difference of 8.9 months and a hazard ratio (HR) ranging from 0.25 to 0.73 across cancer types, demonstrating an immortal time bias. One hundred and forty-nine patients received matched therapies in clinical trials on the basis of their identified alterations. Among patients with colorectal cancer harboring actionable alterations, the median OS was significantly longer in patients who received matched therapies in trials than in those who did not (HR, 0.52; 95% CI, 0.26 to 1.01; P = .049). Cancer-specific pathway alterations were significantly associated with shorter survival and related to primary resistance to matched trial therapies., Conclusion: Our genomic profiling program led to patient enrollment in targeted clinical trials and improved survival of patients with colorectal cancer who received matched therapies in clinical trials. To avoid immortal time bias, precautions are needed when using data from patients who have undergone NGS testing after initiation of the evaluated treatment line.

Background and Objective The periodontal ligament ( PDL) is characterized by rapid turnover, high remodeling capacity and high inherent regenerative potential compared with other connective tissues. Periostin, which is highly expressed in the fibroblasts in the PDL, has been widely discussed in relation to collagen fibrillogenesis in the PDL. Recently, several reports have indicated periostin in cell migration. The aim of this study was to examine whether human PDL fibroblasts ( hPDLFs) with high levels of periostin expression promote the migration of human bone marrow mesenchymal stem cells ( hMSCs). Material and Methods The migration of hMSCs was examined by transwell chamber migration assay under different conditions: medium alone, hPDLFs, human dermal fibroblasts, recombinant periostin, integrin αvβ3 blocking antibody (anti- CD51/61 antibody) and inhibitors of FAK ( PF431396) and PI3 K ( LY294002). Phosphorylation of FAK and Akt in hMSCs under stimulation of periostin was examined by western blotting. Results The migration assay revealed that the number of migrated hMSCs by hPDLFs was significantly larger than those by dermal fibroblasts, periostin small interfering RNA hPDLFs and medium alone. Furthermore, recombinant periostin also strongly induced hMSC migration. The addition of anti- CD51/61 antibody, PF431396 and LY294002 caused a significant reduction in the number of migrated hMSCs respectively. The anti- CD51/61 antibody inhibited both FAK and Akt phosphorylations under periostin stimulation. PF431396 inhibited both FAK and Akt phosphorylations. LY294002 inhibited only Akt phosphorylation, and FAK phosphorylation was not influenced under periostin stimulation. Conclusion Periostin expression in hPDLFs promotes the migration of hMSCs through the αvβ3 integrin/ FAK/ PI3 K/ Akt pathway in vitro. [ABSTRACT FROM AUTHOR]

Maoto, a traditional Japanese medicine (Kampo), is widely used to treat upper respiratory tract infections, including influenza virus infection. Although maoto is known to inhibit pro-inflammatory responses in a rodent model of acute inflammation, its underlying mechanism remains to be determined. In this study, we investigated the involvement of immune responses and noradrenergic function in the inhibitory action of maoto. In a mouse model of polyI:C-induced acute inflammation, maoto was administered orally in conjunction with intraperitoneal injection of PolyI:C (6 mg/kg), and blood was collected after 2 h for measurement of plasma cytokines by ELISA. Maoto significantly decreased PolyI:C-induced TNF-α levels and increased IL-10 production. Neither pretreatment with IL-10 neutralizing antibodies nor T-cell deficiency using nude mice modified the inhibitory effect of maoto, indicating that the anti-inflammatory effects of maoto are independent of IL-10 and T cells. Furthermore, the inhibitory effects of maoto on PolyI:C-induced TNF-α production were not observed in ex vivo splenocytes, suggesting that maoto does not act directly on inflammatory cells. Lastly, pretreatment with a β-adrenergic receptor antagonist partially cancelled the anti-inflammatory effects of maoto. Collectively, these results suggest that maoto mediates its anti-inflammatory effects via β-adrenergic receptors in vivo., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Previously, we isolated and identified anti-mutagenic and anti-inflammatory components from Vitis coignetiae (crimson glory vine, known as yamabudo in Japan) as 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid. We also reported that the oral intake of a partially purified fraction from yamabudo juice (yamabudo-fr) or DBQ affords significant protection against two-stage skin carcinogenesis in mice. In this study, we found that oral intake of yamabudo-fr or DBQ affords significant protection against a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse model of lung tumorigenesis. Furthermore, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ. NNK is known to be a DNA-methylating and alkylating agent; thus, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ in relation to DNA methylation. Pretreatment with yamabudo-fr or DBQ dose-dependently decreased formation of O 6-methylguanine and N 7-methylguanine in DNA of the A549 human lung epithelial-like cell line treated with a methylating agent, 1-methyl-3-nitro-1-nitrosoguanidine. Yamabudo juice and DBQ inhibited the mutagenicity of NNK in the Ames test using Salmonella typhimurium TA1535 but not S. typhimurium YG7108, an alkylguanine DNA alkyltransferase-deficient strain (same as TA1535 but Δada st ::Km r , Δogt st ::Cm r ). Yamabudo juice and DBQ might accelerate the repair of DNA damage caused by NNK and reduce DNA damage to cells. We also investigated the effects of yamabudo juice and DBQ on signaling pathways in A549 cells. With or without epidermal growth factor stimulation, phosphorylation of Erk1/2, Akt and Stat3 in A549 cells was significantly decreased in the presence of yamabudo juice or DBQ, indicating that yamabudo juice and DBQ suppressed PI3K/AKT, MAPK/ERK and JAK/STAT3 signaling pathways. These results suggest that both initiation and growth/progression steps in carcinogenesis, especially anti-oxidant effects, stimulation of repair of alkyl DNA adducts and suppressed growth signaling pathways are potential anti-tumorigenic targets of yamabudo juice and DBQ in NNK-induced lung tumorigenesis. • Yamabudo juice and 2,6-dimethoxy-1,4-benzoquinone (DBQ) exhibited chemopreventive effects in NKK-induced lung tumors in mice. • Yamabudo juice and DBQ decreased methyl DNA adducts and stimulated DNA repair. • Yamabudo juice and DBQ suppressed growth signaling of Akt, Erk and Stat3 in A549 cells. [ABSTRACT FROM AUTHOR]

Amlexanox, a small molecule targeted therapy which has been used in the treatment of atopic conditions was previously but is not currently available in the United States. Amlexanox has also been legally utilized and administered in Japan as a treatment for asthma, a chronic pulmonary disease characterized by inflammation of the lower respiratory tract. Amlexanox's immune modulatory effects have been the subject of studies which have repurposed the drug for potential therapeutic applications in metabolic and inflammatory disease. Because amlexanox inhibits TANK-binding kinase1 (TBK1) and nuclear factor kB kinase epsilon (IKKε), several studies have demonstrated its usefulness through its evidence downregulation of the immune system and attenuation of downstream TBK1 signaling. Novel therapies, such as amlexanox, for inflammatory conditions such as asthma will continue to be of value in clinical management. This report summarizes key applications of the drug based on animal and human studies and explores its potential in treatment of metabolic and inflammatory diseases., (Copyright ©2020, Yale Journal of Biology and Medicine.)

Despite advances in the treatment of hormone receptor-positive, HER2- metastatic breast cancer, the disease is rarely curable. In this review, we focus on the use of CDK4/6 inhibitors, examining clinical experience and the mechanisms underlying the development of resistance, and evaluating treatment options after failure to respond to CDK4/6 inhibitors. Current basic research supports the use of mammalian target of rapamycin inhibitors after CDK4/6 inhibitor failure; however, more data are needed, particularly regarding treatment sequencing. Real-world data studies may help to fill the current knowledge gap, particularly where large-scale randomized controlled studies are not feasible.

Background: Resistance to first-generation or second-generation EGFR tyrosine kinase inhibitor (TKI) monotherapy develops in almost half of patients with EGFR-positive non-small-cell lung cancer (NSCLC) after 1 year of treatment. The JO25567 phase 2 trial comparing erlotinib plus bevacizumab combination therapy with erlotinib monotherapy established the activity and manageable toxicity of erlotinib plus bevacizumab in patients with NSCLC. We did a phase 3 trial to validate the results of the JO25567 study and report here the results from the preplanned interim analysis., Methods: In this prespecified interim analysis of the randomised, open-label, phase 3 NEJ026 trial, we recruited patients with stage IIIB-IV disease or recurrent, cytologically or histologically confirmed non-squamous NSCLC with activating EGFR genomic aberrations from 69 centres across Japan. Eligible patients were at least 20 years old, and had an Eastern Cooperative Oncology Group performance status of 2 or lower, no previous chemotherapy for advanced disease, and one or more measurable lesions based on Response Evaluation Criteria in Solid Tumours (1.1). Patients were randomly assigned (1:1) to receive oral erlotinib 150 mg per day plus intravenous bevacizumab 15 mg/kg once every 21 days, or erlotinib 150 mg per day monotherapy. Randomisation was done by minimisation, stratified by sex, smoking status, clinical stage, and EGFR mutation subtype. The primary endpoint was progression-free survival. This study is ongoing; the data cutoff for this prespecified interim analysis was Sept 21, 2017. Efficacy was analysed in the modified intention-to-treat population, which included all randomly assigned patients who received at least one dose of treatment and had at least one response evaluation. Safety was analysed in all patients who received at least one dose of study drug. The trial is registered with the University Hospital Medical Information Network Clinical Trials Registry, number UMIN000017069., Findings: Between June 3, 2015, and Aug 31, 2016, 228 patients were randomly assigned to receive erlotinib plus bevacizumab (n=114) or erlotinib alone (n=114). 112 patients in each group were evaluable for efficacy, and safety was evaluated in 112 patients in the combination therapy group and 114 in the monotherapy group. Median follow-up was 12·4 months (IQR 7·0-15·7). At the time of interim analysis, median progression-free survival for patients in the erlotinib plus bevacizumab group was 16·9 months (95% CI 14·2-21·0) compared with 13·3 months (11·1-15·3) for patients in the erlotinib group (hazard ratio 0·605, 95% CI 0·417-0·877; p=0·016). 98 (88%) of 112 patients in the erlotinib plus bevacizumab group and 53 (46%) of 114 patients in the erlotinib alone group had grade 3 or worse adverse events. The most common grade 3-4 adverse event was rash (23 [21%] of 112 patients in the erlotinib plus bevacizumab group vs 24 [21%] of 114 patients in the erlotinib alone group). Nine (8%) of 112 patients in the erlotinib plus bevacizumab group and five (4%) of 114 patients in the erlotinib alone group had serious adverse events. The most common serious adverse events were grade 4 neutropenia (two [2%] of 112 patients in the erlotinib plus bevacizumab group) and grade 4 hepatic dysfunction (one [1%] of 112 patients in the erlotinib plus bevacizumab group and one [1%] of 114 patients in the erlotinib alone group). No treatment-related deaths occurred., Interpretation: The results of this interim analysis showed that bevacizumab plus erlotinib combination therapy improves progression-free survival compared with erlotinib alone in patients with EGFR-positive NSCLC. Future studies with longer follow-up, and overall survival and quality-of-life data will be required to further assess the efficacy of this combination in this setting., Funding: Chugai Pharmaceutical., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: To examine the influence of smoking on biologics treatment against different therapeutic targets, such as TNFα, IL-6, and T cell, in rheumatoid arthritis (RA) and elucidate the underlying molecular mechanism., Methods: The association between drug-discontinuation due to poor therapeutic response and smoking status was analyzed individually in biologics against different therapeutic targets by a multivariable logistic regression analysis using the "NinJa" Registry, one of the largest cohorts of Japanese RA patients. In vitro enhancement of TNFα-induced NF-κB activation and subsequent proinflammatory cytokine production by cigarette chemical components was examined by RT-PCR, qPCR, ELISA, and western blotting using an immortalized rheumatoid synovial cell line, MH7A., Results: The rate of drug-discontinuation due to poor therapeutic response was higher in the current smoking group than in the never- or ever-smoking groups (the odds ratio of current/never smoking: 2.189, 95%CI; 1.305-3.672,P = 0.003; current/ever: 1.580, 95%CI; 0.879-2.839,P = 0.126) in the TNF inhibitor (TNFi) treatment group. However, this tendency was not observed in either the IL-6 or T cell inhibitor treatment groups. Cigarette smoke chemical components, such as benzo[α]pyrene, known as aryl hydrocarbon receptor (AhR) ligands, themselves activated NF-κB and induced proinflammatory cytokines, IL-1β and IL-6. Furthermore, they also significantly enhanced TNFα-induced NF-κB activation and proinflammatory cytokine production. This enhancement was dominantly inhibited by Bay 11-7082, an NF-κB inhibitor., Conclusions: These results suggest a crosstalk between TNFα signaling and AhR signaling in NF-κB activation which may constitute one of the molecular mechanisms underlying the higher incidence of drug-discontinuation in RA patients undergoing TNFi treatment with smoking habits., (Copyright © 2018. Published by Elsevier Ltd.)

Background: Pathophysiology of type 1 autoimmune pancreatitis (AIP) is still unclear. We previously reported that M2 macrophages might play an important role in type 1 AIP. Recently, it has been reported that basophils regulate differentiation to M2 macrophages. In this study, we investigated basophils from the pancreatic tissue and peripheral blood of individuals with type 1 AIP., Methods: By using immunohistochemistry, we investigated basophils in pancreatic tissue from 13 patients with type 1 AIP and examined expression of toll-like receptors (TLRs) by these cells. Additionally, we obtained peripheral blood samples from 27 healthy subjects, 40 patients with type 1 AIP, 8 patients with alcoholic chronic pancreatitis, 10 patients with bronchial asthma, and 10 patients with atopic dermatitis, and analyzed activation of basophils by stimulating them with ligands of TLR1-9. We also compared TLR expression in basophils from the tissue and blood samples., Results: Basophils were detected in pancreatic tissues from 10 of 13 patients with type 1 AIP. Flow cytometric analysis revealed that the ratios of basophils activated by TLR4 stimulation in type 1 AIP (9.875 ± 1.148%) and atopic dermatitis (11.768 ± 1.899%) were significantly higher than those in healthy subjects (5.051 ± 0.730%; P < 0.05). Levels of basophils activated by TLR2 stimulation were higher in seven type 1 AIP cases. Furthermore, stimulation of TLR2 and/or TLR4, which were expressed by basophils in pancreas, activated basophils in peripheral blood., Conclusions: Basophils activated via TLR signaling may play an important role in the pathophysiology of type 1 AIP.

Coincident with the blossoming of the sakura was the 14th annual CDB Symposium hosted by the RIKEN Center for Developmental Biology in Kobe, Japan. This year's meeting, 'Size in Development: Growth, Shape and Allometry' focused on the molecular and cellular mechanisms underlying differences in size and shape and how they have evolved. On display was the power of using diverse approaches ranging from the study of organoids to whole organisms., (© 2016. Published by The Company of Biologists Ltd.)

Behçet's disease (BD) is a multi-system inflammatory disorder of unknown etiology. Two recent genome-wide association studies (GWASs) of BD confirmed a strong association with the MHC class I region and identified two non-HLA common genetic variations. In complex diseases, multiple factors may target different sets of genes in the same pathway and thus may cause the same disease phenotype. We therefore hypothesized that identification of disease-associated pathways is critical to elucidate mechanisms underlying BD, and those pathways may be conserved within and across populations. To identify the disease-associated pathways, we developed a novel methodology that combines nominally significant evidence of genetic association with current knowledge of biochemical pathways, protein-protein interaction networks, and functional information of selected SNPs. Using this methodology, we searched for the disease-related pathways in two BD GWASs in Turkish and Japanese case-control groups. We found that 6 of the top 10 identified pathways in both populations were overlapping, even though there were few significantly conserved SNPs/genes within and between populations. The probability of random occurrence of such an event was 2.24E-39. These shared pathways were focal adhesion, MAPK signaling, TGF-β signaling, ECM-receptor interaction, complement and coagulation cascades, and proteasome pathways. Even though each individual has a unique combination of factors involved in their disease development, the targeted pathways are expected to be mostly the same. Hence, the identification of shared pathways between the Turkish and the Japanese patients using GWAS data may help further elucidate the inflammatory mechanisms in BD pathogenesis.

Global changes in gene expression accompany the development of cancer. Thus, inherited variants in miRNA-binding sites are likely candidates for conferring inherited susceptibility. Using an in silico approach, we compiled a comprehensive list of SNPs predicted to modulate miRNA binding in genes from several key lung cancer pathways. We then investigated whether these SNPs were associated with lung cancer risk in two independent populations. In general, SNPs in miRNA-binding sites are rare. However, some allelic variation was observed. We found that rs1126579 in CXCR2 was associated with a reduced risk of lung cancer in both European American [ORTT vs. CC 0.56 (0.37-0.88); P = 0.008] and Japanese [ORTT vs. CC 0.62 (0.38-1.00); P = 0.049] populations. Furthermore, we found that the SNP disrupted a novel binding site for miR-516a-3p, led to a moderate increase in CXCR2 mRNA and protein expression, and increased MAPK signaling. Moreover, analysis of rs1126579 with serum levels of IL8, its endogenous ligand, supported an interaction whereby rs1126579-T and high serum IL8 conferred synergistic protection from lung cancer. Our findings demonstrate a function for a 3'UTR SNP in modulating CXCR2 expression, signaling, and susceptibility to lung cancer., (©2014 American Association for Cancer Research.)

In Western countries, gene alterations involving the CRLF2-JAK signaling pathway are identified in approximately 50-60% of patients with Down syndrome-associated acute lymphoblastic leukemia (DS-ALL), and this pathway is considered a potential therapeutic target. The frequency of BTG1 deletions in DS-ALL is controversial. IKZF1 deletions, found in 20-30% of DS-ALL patients, are associated with a poor outcome and EBF1 deletions are very rare (∼2%). We analyzed 38 patients to determine the frequencies and clinical implications of CRLF2-JAK pathway genetic alterations and recurrent gene deletions in Japanese DS-ALL patients. We confirmed a high incidence of P2RY8-CRLF2 (29%) and JAK2 mutations (16%), though the frequency of P2RY8-CRLF2 was slightly lower than that in Western countries (∼50%). BTG1 deletions were common in our cohort (25%). IKZF1 deletions were detected in 25% of patients and associated with shorter overall survival (OS). EBF1 deletions were found at an unexpectedly high frequency (16%), and at a significantly higher level in P2RY8-CRLF2-positive patients than in P2RY8-CRLF2-negative patients (44% vs. 4%, P=0.015). Deletions of CDKN2A/B and PAX5 were common in P2RY8-CRLF2-negative patients (48 and 39%, respectively) but not in P2RY8-CRLF2-positive patients (11% each). Associations between these genetic alterations and clinical characteristics were not observed except for inferior OS in patients with IKZF1 deletions. These results suggest that differences exist between the genetic profiles of DS-ALL patients in Japan and in Western countries, and that P2RY8-CRLF2 and EBF1 deletions may cooperate in leukemogenesis in a subset of Japanese DS-ALL patients., (© 2014 Wiley Periodicals, Inc.)

The access of bone morphogenetic protein (BMP) to the BMP receptors on the cell surface is regulated by its antagonist noggin, which binds to heparan-sulfate proteoglycans on the cell surface. Noggin is encoded by NOG and mutations in the gene are associated with aberrant skeletal formation, such as in the autosomal dominant disorders proximal symphalangism (SYM1), multiple synostoses syndrome, Teunissen-Cremers syndrome, and tarsal-carpal coalition syndrome. NOG mutations affecting a specific function may produce a distinct phenotype. In this study, we investigated a Japanese pedigree with SYM1 and conductive hearing loss and found that it carried a novel heterozygous missense mutation of NOG (c.406C>T; p.R136C) affecting the heparin-binding site of noggin. As no mutations of the heparin-binding site of noggin have previously been reported, we investigated the crystal structure of wild-type noggin to investigate molecular mechanism of the p.R136C mutation. We found that the positively charged arginine at position 136 was predicted to be important for binding to the negatively charged heparan-sulfate proteoglycan (HSPG). An in silico docking analysis showed that one of the salt bridges between noggin and heparin disappeared following the replacement of the arginine with a non-charged cysteine. We propose that the decreased binding affinity of NOG with the p.R136C mutation to HSPG leads to an excess of BMP signaling and underlies the SYM1 and conductive hearing loss phenotype of carriers., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In humans, narcolepsy with cataplexy (narcolepsy) is a sleep disorder that is characterized by sleepiness, cataplexy and rapid eye movement (REM) sleep abnormalities. Narcolepsy is caused by a reduction in the number of neurons that produce hypocretin (orexin) neuropeptide. Both genetic and environmental factors contribute to the development of narcolepsy.Rare and large copy number variations (CNVs) reportedly play a role in the etiology of a number of neuropsychiatric disorders. Narcolepsy is considered a neurological disorder; therefore, we sought to investigate any possible association between rare and large CNVs and human narcolepsy. We used DNA microarray data and a CNV detection software application, PennCNV-Affy, to detect CNVs in 426 Japanese narcoleptic patients and 562 healthy individuals. Overall, we found a significant enrichment of rare and large CNVs (frequency ≤1%, size ≥100 kb) in the patients (case-control ratio of CNV count=1.54, P=5.00 × 10(-4)). Next, we extended a region-based association analysis by including CNVs with its size ≥30 kb. Rare and large CNVs in PARK2 region showed a significant association with narcolepsy. Four patients were assessed to carry duplications of the gene region, whereas no controls carried the duplication, which was further confirmed by quantitative PCR assay. This duplication was also found in 2 essential hypersomnia (EHS) patients out of 171 patients. Furthermore, a pathway analysis revealed enrichments of gene disruptions by rare and large CNVs in immune response, acetyltransferase activity, cell cycle regulation and regulation of cell development. This study constitutes the first report on the risk association between multiple rare and large CNVs and the pathogenesis of narcolepsy. In the future, replication studies are needed to confirm the associations.

Objective: The objective of this study was to investigate the significance of the activated Akt-mammalian target of rapamycin (Akt-mTOR) signaling pathway in the progression of prostate cancer., Materials and Methods: The expression levels of Akt, phosphorylated Akt (p-Akt), mTOR and phosphorylated mTOR (p-mTOR) in 175 prostate specimens, including 61 normal prostate tissues as a control, 24 high-grade prostatic intraepithelial neoplasias (HGPINs) and 90 clinically localized prostate cancers, were evaluated by immunohistochemical staining. p-Akt and p-mTOR ratios, which were defined as the expression level of p-Akt in relation to that of Akt and the expression level of p-mTOR in relation to that of mTOR, respectively, in these specimens were calculated., Results: Expression levels of all four molecules, including Akt, p-Akt, mTOR and p-mTOR, were significantly greater in the HGPIN group compared with the normal control and prostate cancer groups. Furthermore, the p-Akt ratio in the prostate cancer group was significantly lower than that in the HGPIN group, while there was no significant difference in the p-mTOR ratio between the HGPIN and prostate cancer groups. In the prostate cancer group, no significant relationships were observed between major clinicopathological parameters and the expression levels as well as the ratios of p-Akt or p-mTOR., Conclusions: The Akt-mTOR signaling pathway may play a limited role in the progression of prostate cancer., (2014 S. Karger AG, Basel.)

6-(methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi (Wasabia japonica), which is a popular spice in Japan. 6-MSITC has been reported to inhibit the proliferation of breast cancer and melanoma cell lines. We inoculated 30 female Balb-nu/nu mice with MDA-MB-231 or -453 cells, and orally administered varying concentrations of 6-MSITC for 12 days following tumor growth. The tumor volumes and tumor weights from mice inoculated with MDA-MB-231 cells, and the tumor volumes of MDA-MB-453 cells were significantly inhibited by 6-MSITC on Days 9 and 11 after drug administration. DNA fragmentation, DNA ladder, and caspase 3/7 activity performed in vitro revealed that 6-MSITC induced apoptosis of MDA-MB-231, MDA-MB-453, and MCF-7 cells. Furthermore, nuclear factor-κB (NF-κB) expression in the nuclei and phosphorylation of inhibitor κBα (IκBα) was downregulated by 6-MSITC in a concentration-dependent manner; however, this activity was not observed in MCF-7 cells. Moreover, this downregulation of phosphorylated IκBα by 6-MSITC in MDA-MB-231 and -453 cells supports its inhibitory effects on NF-κB activity. The expression of phosphorylated AKT (pAKT) reduced by 6-MSITC was confirmed in MDA-MB-231 cells. Thus, we conclude that 6-MITC promotes apoptosis of breast cancer cells by inhibiting NF-kB and therefore releasing its control of the PI3K/AKT pathway.

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes in the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies., (© 2013.)

Gastric neoplasia of chief cell-predominant type (GN-CCP) has been reported as a new, rare variant of gastric tumor. GN-CCPs were defined as tumors consisting of irregular anastomosing glands of columnar cells mimicking chief cells of fundic gland with nuclear atypia and prolapse-type submucosal involvement. We comparatively evaluated clinicopathologic features between 31 GN-CCPs and 130 cases of conventional gastric adenocarcinoma invading into submucosa (CGA-SM) in addition to nuclear β-catenin immunolabeling and direct sequencing of members of the Wnt/β-catenin pathway, CTNNB1, APC, and AXIN, in a subset of these tumors. GN-CCP presented as small protruded lesions located in the upper third of the stomach, with minimal involvement into the submucosa and rare lymphovascular invasion. None of the lesions have demonstrated a recurrence of disease or metastasis on follow-up. Nuclear β-catenin immunolabeling was higher in GN-CCP (labeling index [LI]: median, 19.3%; high expresser [LI >30%], 7/27 cases [26%]) than CGA-SM (median LI, 14.7%; high expresser, 1/19 cases [6%]). Missense mutation of APC was observed in 1 GN-CCP but not CGA-SM. Missense or nonsense mutations of CTNNB1 and AXIN1 were higher in GN-CCPs (14.8%, both) than CGA-SMs (5.3%, both). Missense mutations of AXIN2 were higher in GN-CCPs (25.9%) than in CGA-SMs (10.5%). Overall, 14 (51.9%) of 27 GN-CCPs and 5 (26.3%) of 19 CGA-SM cases harbored at least 1 of these gene mutations. In conclusion, GN-CCPs as a unique variant of nonaggressive tumor are characterized by nuclear β-catenin accumulation and mutation of CTNNB1 or AXIN gene, suggesting activation of the Wnt/β-catenin pathway., (© 2013.)

The Targeted Proteins Research Program (TPRP) promoted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan is the phase II of structural biology project (2007-2011) following the Protein 3000 Project (2002-2006) in Japan. While the phase I Protein 3000 Project put partial emphasis on the construction and maintenance of pipelines for structural analyses, the TPRP is dedicated to revealing the structures and functions of the targeted proteins that have great importance in both basic research and industrial applications. To pursue this objective, 35 Targeted Proteins (TP) Projects selected in the three areas of fundamental biology, medicine and pharmacology, and food and environment are tightly collaborated with 10 Advanced Technology (AT) Projects in the four fields of protein production, structural analyses, chemical library and screening, and information platform. Here, the outlines and achievements of the 35 TP Projects are summarized in the system named TP Atlas. Progress in the diversified areas is described in the modules of Graphical Summary, General Summary, Tabular Summary, and Structure Gallery of the TP Atlas in the standard and unified format. Advances in TP Projects owing to novel technologies stemmed from AT Projects and collaborative research among TP Projects are illustrated as a hallmark of the Program. The TP Atlas can be accessed at http://net.genes.nig.ac.jp/tpatlas/index&#95;e.html .

Rheumatoid arthritis is an autoimmune disease with a complex etiology, leading to inflammation of synovial tissue and joint destruction. Through a genome-wide association study (GWAS) and two replication studies in the Japanese population (7,907 cases and 35,362 controls), we identified two gene loci associated with rheumatoid arthritis susceptibility (NFKBIE at 6p21.1, rs2233434, odds ratio (OR) = 1.20, P = 1.3 × 10(-15); RTKN2 at 10q21.2, rs3125734, OR = 1.20, P = 4.6 × 10(-9)). In addition to two functional non-synonymous SNPs in NFKBIE, we identified candidate causal SNPs with regulatory potential in NFKBIE and RTKN2 gene regions by integrating in silico analysis using public genome databases and subsequent in vitro analysis. Both of these genes are known to regulate the NF-κB pathway, and the risk alleles of the genes were implicated in the enhancement of NF-κB activity in our analyses. These results suggest that the NF-κB pathway plays a role in pathogenesis and would be a rational target for treatment of rheumatoid arthritis., Competing Interests: The authors have declared that no competing interests exist.

Cultivated ginseng (CG) (Panax ginseng C.A. Meyer), an herb used in Korean herbal medicine, has been widely used in China and Japan to treat fatigue and to enhance resistance to many diseases. It contains many bioactive constituents, including various ginsenosides that are believed to have antioxidant, immunostimulatory, and antiaging activities. Previous studies have revealed that treatment with Panax ginseng is significantly associated with reduced photoaging, but the underlying mode of action has not been elucidated. In this study, we hypothesized that CG inhibits ultraviolet B (UVB)-induced collagenase activation through mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB)/activator protein-1 (AP-1)-dependent signaling in human skin fibroblasts. HS68 cells were treated with CG, followed by irradiation with UVB. Those effects were assessed by semiquantitative polymerase chain reaction, Western blotting, and enzymic activity assays. We found that CG increased cell viability and inhibited the production of reactive oxygen species in HS68 cells exposed to UVB irradiation. Pretreatment of HS68 cells with CG inhibited UVB-induced production of matrix metalloproteinase (MMP) 1 and MMP-13. Western blot analysis further revealed that CG markedly suppressed the enhancement of collagen degradation in UVB-exposed HS68 cells. Cultivated ginseng also suppressed UVB-induced activation of NF-κB, c-Jun, and c-Fos and the phosphorylation of MAPKs, which are upstream modulators of NF-κB and AP-1. These results indicate that CG inhibits UVB-induced collagenolytic MMP production by interfering with MAPK/AP-1 and NF-κB signaling and thus may be useful in the prevention and treatment of skin photoaging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although TNF inhibitors have dramatically improved the outcome of patients with rheumatoid arthritis, 30-40% of patients do not respond well to them and treatment needs to be changed. In an effort to discriminate good and poor responders, we focused on the change in serum and synovial fluid levels of interleukin (IL-) 33 before and after treatment with TNF inhibitors. They were also measured in synovial fluids from 17 TNF inhibitor-naïve patients, and fibroblast-like synoviocytes (FLS) in-culture from 6 patients and correlated with various pro-inflammatory cytokines. Serum levels of IL-33 at 6 months after treatment decreased significantly in responders, while they did not change in non-responders. Synovial fluid levels of IL-33 in 6 patients under treatment with TNF inhibitors stayed high in 3 who were refractory and slightly elevated in 2 moderate responders, while they were undetectable in one patient under remission. Among inflammatory cytokines measured in 17 synovial fluids from TNF inhibitor-naïve patients, levels of IL-33 showed a significant positive correlation only to those of IL-1β. IL-1β increased IL-33 expression markedly in FLS in vitro, compared to TNF-α. IL-1β might be inducing RA inflammation through producing pro-inflammatory IL-33 in TNF inhibitor-hypo-responders. Sustained elevation of serum and/or synovial levels of IL-33 may account for a poor response to TNF inhibitors, although how TNF inhibitors affect the level of IL-33 remains to be elucidated.

Peptide signaling plays a major role in various aspects of plant growth and development, as has been shown in recent biochemical, genetic and bioinformatic studies. There are over a dozen secreted peptides recognized in plants known to regulate cellular functions. To become functional, these secreted peptide signals often undergo post-translational modifications, such as tyrosine sulfation, proline hydroxylation, and hydroxyproline arabinosylation, and successive proteolytic processing. These types of ‘small post-translationally modified peptide signals’ are one of the major groups of peptide signals found in plants. In parallel with the discovery of peptide signals, specific receptors for such peptide signals were identified as being membrane-localized leucine-rich repeat receptor kinases. This short review highlights the recent progress in research on small post-translationally modified peptide signals, including our own research.

The insulin-like growth factor (IGF) signaling system plays a central role in cellular growth, differentiation and proliferation. Although the association between IGF1 gene polymorphisms and cancer risk has been evaluated for several carcinomas, this association has not yet been examined for stomach cancer. We investigated the association between IGF1 polymorphisms and the risk of stomach cancer in a Japanese population. A total of 703 patients with stomach cancer and 1462 non-cancer control subjects were enrolled in this case-control study. Associations between polymorphisms of 10 IGF1 loci and the risk of stomach cancer were evaluated using odds ratios (OR) and 95% confidence intervals (CI) in multiple logistic regression models. We observed that the C allele in rs1520220 and the G allele in rs4764887 were significantly associated with stomach cancer risk in the per-allele model after adjusting for other risk factors (OR: 1.14 [95% CI: 1.00-1.30] and OR: 1.18 [95% CI: 1.02-1.36], respectively). We also observed a positive and dose-dependent association between the number of risk alleles and stomach cancer risk (P-trend: 0.019) when examining the two loci in the same model. These associations were still seen after adjusting for potential confounders, including sex, age, smoking status, history of diabetes and family history of stomach cancer. We did not find any significant interaction between these factors and the number of risk alleles. In conclusion, we observed a significant association between IGF1 polymorphisms and stomach cancer risk among a Japanese population. Examination of the biological significance of IGF1 is warranted., (© 2011 Japanese Cancer Association.)

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signaling molecules by many Gram-negative bacteria. Here, 413 bacterial strains were obtained from the roots of potato plants and screened for AHL-degrading bacteria using Chromobacterium violaceum reporter strains. Sixty one isolates degraded N-hexanoyl-L-homoserine lactone (C6-HSL) within 24 h. Based on their 16S rRNA gene sequences, most of these isolates were assigned to the genus Chryseobacterium and divided into eight groups. Most of the strains degraded N-decanoyl-L-homoserine lactone (C10-HSL) and were active against 3-oxo-substituted AHLs. Groups V and VI were more effective at inactivating AHLs. One group V strain, StRB402, showed putative AHL-lactonase activity. This is the first report of AHL-degrading Chryseobacterium strains.

Purpose: The hedgehog (Hh) signaling pathway is activated in some adult cancers. On the other hand, the Hh signaling pathway plays an important role in the development of the neural crest in embryos. The aim of this study is to show the activation of Hh signaling pathway in neuroblastoma (NB), a pediatric malignancy arising from neural crest cells, and to reveal the meaning of the Hh signaling pathway in NB development., Methods: This study analyzed the expression of Sonic hedgehog (Shh), GLI1, and Patched 1 (Ptch1), transactivators of Hh signaling pathway, by immunohistochemistry in 82 NB and 10 ganglioneuroblastoma cases. All 92 cases were evaluated for the status of MYCN amplification., Results: Of the 92 cases, 67 (73%) were positive for Shh, 62 cases (67%) were positive for GLI1, and 73 cases (79%) were positive for Ptch1. Only 2 (10%) of the 20 cases with MYCN amplification were positive for Shh and GLI1, and 4 cases (20%) were positive for Ptch1 (MYCN amplification vs no MYCN amplification, P ≦ .01). The percentage of GLI1-positive cells in the cases with INSS stage 1 without MYCN amplification was significantly higher than that with INSS stage 4. Of 72 cases without MYCN amplification, 60 were GLI1-positive. Twelve cases were GLI1-negative, and the prognosis of the GLI1-positive cases was significantly better than that of the GLI1-negative cases (P = .015)., Conclusions: Most of NBs without MYCN amplification were positive for Shh, GLI1, and Ptch1. In the cases without MYCN amplification, the high expression of GLI1 was significantly associated with early clinical stage and a good prognosis of the patients. In contrast to adult cancers, the activation of the Hh signaling pathway in NB may be associated with the differentiation of the NB., (Copyright © 2010 Elsevier Inc. All rights reserved.)

I have devoted my life to NMR research for nearly 40 years since I graduated from Tokyo University. Owing to innovative technical developments such as introduction of superconductive magnets into NMR, and development of FT-NMR and two-dimensional NMR, I have spent an interesting and inspired research life. When I look back on my life, I found myself starting from structural chemistry and doing research from the beginning of and all the way to the maturation of structural biology. I have been enjoying scientific challenges with my students and collaborators and I am proud that many young scientists grew up in my laboratory.

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by a single S-locus with multi allelic variety. The male S determinant, SP11/SCR (S-locus protein 11/S-locus cysteine-rich protein), is a small cysteine-rich protein, and the female S determinant, SRK (S-locus receptor kinase), functions as a receptor for SP11 at the surface of stigma papilla cells. Although a few of the following downstream factors in the SP11-SRK signaling cascade have been identified, a comprehensive understanding of the SI mechanism still remains unexplained in Brassicaceae. Analysis of self-compatible (SC) mutants is significant for understanding the molecular mechanism in SI reactions, thus we screened SC lines from a variety of Japanese bulk-populations of B. rapa vegetables. Two lines, TSC4 and TSC28, seem to have disruptions in the SI signaling cascade, while the other line, TSC2, seems to have a deficiency in a female S determinant, SRK. In TSC4 and TSC28, known SI-related factors, i.e. SRK, SP11, MLPK (M-locus protein kinase), THL (thioredoxin-h-like), and ARC1 (arm repeat containing 1), were expressed normally, and their expression levels were comparable with those in SI lines. On a B. rapa genetic linkage map, potential SC genes in TSC4 and TSC28 were mapped on linkage groups A3 and A1, respectively, whereas MLPK, ARC1, and THL were mapped on A3, A4, and A6, respectively. Although potential SC genes of TSC4 and MLPK were on the same linkage group, their positions were apparently independent. These results indicate that the SC genes of TSC4 and TSC28 are independent from the S-locus or known SI-related genes. Thus, the SC lines selected here have mutations in novel factors of the SI signaling cascade, and they will contribute to fill pieces in a signal transduction pathway of the SI system in Brassicaceae.

The effects of chronic administration of Sake (Japanese alcoholic beverage, Nihonshu) on brain and liver of female F334 (Fisher) rats were surveyed via global omic analyses using DNA microarray, 2-DE, and proton nuclear magnetic resonance. Rats weaned at 4 wk of age were given free access to Sake (15% alcohol), instead of water. At 13 months of age, and 24 h after withdrawal of Sake supply, rats were sacrificed, and the whole brain and liver tissues dissected for analyses. In general, molecular changes in brain were found to be less than those in liver. Transcriptomics data revealed 36 and 9, and 80 and 62 up- and down-regulated genes, in the brain and liver, respectively, with binding and catalytic activity gene categories the most prominently changed. Results suggested Sake-induced fragility of brain and liver toxicity/damage, though no significant abnormalities in growth were seen. At protein level, a striking decrease was found in the expression of NADH dehydrogenase (ubiquinone) Fe-S protein 1 in brain, suggesting attenuation of mitochondrial metabolism. In liver, results again suggested an attenuation of mitochondrial function and, in addition, glycoproteins with unknown function were induced at protein and gene levels, suggesting possible changes in glycoprotein binding in that organ. Metabolomic analysis of brain revealed significant increases in valine, arginine/ornithine, alanine, glutamine, and choline with decreases in isoleucine, N-acetyl aspartate, taurine, glutamate, and gamma aminobutyric acid. Our results provide a detailed inventory of molecular components of both brain and liver after Sake intake, and may help to better understand effects of chronic Sake drinking.

The LKB1-AMPK-TORC2 signaling pathway controls glucose homeostasis in the liver, and mediates therapeutic effects of insulin-sensitizing antidiabetic agents. To examine whether genetic variations in genes encoding components of this signaling pathway contribute to increased susceptibility to type 2 diabetes, we screened STK11 (LKB1) and CRTC2 (TORC2) genes for genetic variants and conducted a case-control study in 1787 unrelated Japanese individuals. Additionally, the previously described association between the PRKAA2 (AMPK alpha2-subunit) haplotype and type 2 diabetes was tested for replication. We observed associations of nominal significance with two SNPs, an intronic SNP in the STK11 (rs741765; OR 1.33, 95% CI 1.05-1.67, p=0.017, under a recessive genetic model), and a non-synonymous SNP in the CRTC2 (6909C>T: Arg379Cys; OR 3.01, 95% CI 1.18-7.66, p=0.016, under a dominant model), although neither withstood correction for multiple testing. We were unable to replicate the association between the PRKAA2 haplotype and type 2 diabetes: however, in the single SNP evaluation, an intronic PRKAA2 SNP (rs1418442) that had previously been reported to be associated with serum cholesterol levels in Caucasian females showed a weak association (OR 0.62, 95% CI 0.40-0.96, p=0.030, under a recessive model). Among the three genes investigated herein, gene-gene (SNP-SNP) interaction studies provided evidence for an interaction between STK11 and CRTC2 influencing susceptibility to type 2 diabetes. Our findings suggest that genetic variants of LKB1-AMPK-TORC2 pathway components may exert a weak influence on the occurrence of type 2 diabetes in Japanese.

Objective: Analyses of families with rheumatoid arthritis (RA) have suggested the presence of a putative susceptibility locus on chromosome 14q21-23. This large population-based genetic association study was undertaken to examine this region., Methods: A 2-stage case-control association study of 950 unrelated Japanese patients with RA and 950 healthy controls was performed using >400 gene-based common single-nucleotide polymorphisms (SNPs)., Results: Multiple SNPs in the PRKCH gene encoding the eta isozyme of protein kinase C (PKCeta) showed significant single-locus disease associations, the most significant being SNP c.427+8134C>T (odds ratio 0.72, 95% confidence interval 0.62-0.83, P = 5.9 x 10(-5)). Each RA-associated SNP was consistently mapped to 3 distinct regions of strong linkage disequilibrium (i.e., linkage disequilibrium or haplotype blocks) in the PRKCH gene locus, suggesting that multiple causal variants influence disease susceptibility. Significant SNPs included a novel common missense polymorphism of the PRKCH gene, V374I (rs2230500), which lies within the ATP-binding site that is highly conserved among PKC superfamily members. In circulating lymphocytes, PRKCH messenger RNA was expressed at higher levels in resting T cells (CD4(+) or CD8(+)) than in B cells (CD19(+)) or monocytes (CD14(+)) and was significantly down-regulated through immune responses., Conclusion: Our results provide evidence of the involvement of PRKCH as a susceptibility gene for RA in the Japanese population. Dysregulation of PKCeta signal transduction pathway(s) may confer increased risk of RA through aberrant T cell-mediated autoimmune responses.

Proinflammatory cytokine gene polymorphisms have been demonstrated to associate with gastric cancer risk, of which IL1B-31T/C and -511C/T changes have been well investigated due to the possibility that they may alter the IL1B transcription. The signal transduction target upon interleukin 1 beta (IL1beta) stimulation, the nuclear factor of kappa B (NFkappaB) activation, supports cancer development, signal transduction in which is mediated by FS-7 cell-associated cell surface antigen (FAS) signaling. Based on recent papers describing the prognostic roles of the polymorphisms and the NFkappaB functions on cancer development, we sought to determine if Japanese gastric cancer patients were affected by the IL1B -31/-511 and FAS-670 polymorphisms. A case-control study was conducted on incident gastric adenocarcinoma patients (n=271) and age-gender frequency-matched control subjects (n=271). We observed strong linkage disequilibrium between the T allele at -511 and the C allele at -31 and between the C allele at -511 and the T allele at -31 in IL1B in both the cases and controls (R (2)=0.94). Neither IL1B-31, -511 nor FAS-670 polymorphisms showed significantly different risks of gastric adenocarcinoma. Though FAS-670 polymorphisms did not show any significant difference, the proportion of subjects with IL1B-31TT (or IL1B-511CC) increased according to stage (trend P=0.019). In particular, subjects with stage IV had a two times higher probability of having either IL1B-31TT (or IL1B-511CC) genotype compared with stage I subjects. These observations suggest that IL1B-31TT and IL1B-511CC are associated with disease progression.

We have established several embryonic stem (ES) cell lines of the cynomolgus monkey. They maintain the normal karyotype and pluripotency in culture for long periods. We obtained government approval and grants to produce human ES cell lines from frozen surplus embryos in April 2002. We have established and characterized three human ES cell lines (KhES-1, KhES-2, KhES-3). We started distribution of human ES cells to other research groups in March 2004. It would be important to produce genetically modified monkey and human ES cells for various purposes. After improvement of the transfection and selection methods, we have produced monkey ES cells with integrated transgenes at efficient and reliable rates. We are also investigating reprogramming of somatic cells into pluripotent stem cells by cell fusion with ES cells. Such reprogramming could be used to produce pluripotent stem cells for each patient without therapeutic cloning, which would raise ethical concerns.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) mediate many pathways including the beta-adrenergic signaling pathway. The C825T polymorphism in the gene coding for the beta3 subunit of G proteins (GNB3) has been shown to be associated with several phenotypes such as hypertension, obesity, and diabetes mellitus comprising the metabolic syndrome. The GNB3 C825T polymorphism may therefore be associated with many atherosclerosis-related phenotypes. On these grounds, we studied the C825T polymorphism in relation to atherosclerosis-related phenotypes in a large Japanese population. Analyses in general linear models showed that T carriers had a significantly wider pulse pressure (P=0.0089) as well as a significantly higher systolic blood pressure (P=0.026). In contrast, analyses in logistic regression models showed that the C825T polymorphism was not significantly associated with each of the four major classical risk factors for cardiovascular and cerebrovascular disease (obesity, hypertension, hypertriglyceridemia, and diabetes mellitus). However, a significantly higher percentage of subjects had none of the four disorders in CC homozygotes than in T carriers (P=0.026). Thus, the C825T polymorphism was significantly associated with clustering of these four risk factors. Although the effect of the gene on each phenotype appears to be weak, considering the combined impact of the effects of the C825T polymorphism on risk factors, the GNB3 gene may be an important gene for human health.

The beta(3)-adrenergic receptor gene (BAR-3) allelic variant (Trp64Arg and Arg64Arg) is correlated with obesity or non-insulin-dependent diabetes mellitus. The mitochondrial NADH dehydrogenase subunit-2 gene (ND2) variant (Mt5178A) is associated with longevity or less susceptibility to adult-onset diseases. The frequencies of both the variants are high among the Japanese population. Cross-sectional analysis of these variants was conducted to determine if they correlated well with life-style-related phenotypes and nutrient intake. The body fat rate in the BAR-3 variant+ND2 variant group was higher than those rates in the BAR-3 normal+ND2 variant, BAR-3 normal+ND2 normal. The BAR-3 normal+ND2 variant group preferred much carbohydrate and less animal protein compared with other three groups. A combination of SNPs of the nuclear BAR-3 and the mitochondrial ND2 genes may affect eating behavior besides the biochemical and metabolic process of signal transduction and electron transfer system.

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that is produced in the platelet, bone, placenta, and other tissues. It acts as a growth inhibitor in many types of cells, and also mediates extracellular matrix production and immunosuppression. Mutations in the specific domain of its gene ( TGFB1) cause Camurati-Engelmann disease, a bone-sclerosing disorder, and those in other domains may be associated with osteoporosis. We identified 106 single-nucleotide polymorphisms and 11 other types of variations in TGFB1and six other genes. These genes were TGF-beta type I receptor gene (TGFBR1), TGF-beta type II receptor gene (TGFBR2), SMAD2 gene (SMAD2), SMAD3 gene (SMAD3), SMAD4 gene (SMAD4), and SMAD7 gene (SMAD7), all of which compose the TGF-beta1 signaling pathway. We also estimated allele frequencies of these DNA polymorphisms among 48 Japanese individuals. Our data will provide a useful resource for the study of disease susceptibility.