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The sonic hedgehog (Shh) pathway is activated in ∼30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes. [Cancer Res 2007;67(8):3871–7]

Supplementary Figure 1 and Tables 1-2 from Medulloblastomas Derived from Cxcr6 Mutant Mice Respond to Treatment with a Smoothened Inhibitor

Background: Human resource management may be difficult for community pharmacists because of recent drastic changes in work volume and content. In Japan, few studies have been conducted on job satisfaction, well-being, and quality of work life (QWL) among community pharmacists. Focusing on QWL, a more comprehensive concept than job satisfaction, this study aimed to develop a multi-trait-based QWL scale for Japanese community pharmacists (QWLS for JCP) and test its reliability and validity. Methods:A questionnaire survey was conducted among 2027 pharmacists working in pharmacies, with the cooperation of 20 corporations running pharmacies. The collected data were subjected to principal component factor analysis with Promax rotation using SPSS28. Results: The following five significant components were identified based on factor analysis using data from 1966 pharmacists. These form the basis of the QWLS for JCP: “Influence of work on mind and body,” “Relationships with colleagues,” “Relationship with the boss,” “Meaning of existence in the workplace,” and “Pride in work.” Cronbach’s alpha, which expresses reliability, was between 0.585 and 0.854 for these subscales. Conclusions: The QWLS for JCP significantly explained the concept of QWL, which means that its validity was sufficient.

Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.

Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.

The establishment and implementation of a healthy lifestyle is fundamental to public health and is an important issue for working-aged people, as it affects not only them but also the future generations. However, due to the COVID-19 pandemic and associated behavioural restrictions, lifestyles have altered, and, in certain environments, significantly worsened. In the present study, we conducted a project to improve the intestinal environment by focussing on the dietary habits of participants, utilising the living laboratory as a social technology to explore how to adapt to this drastic environmental change. We held eight workshops for voluntary participants and implemented a self-monitoring process of recording dietary behaviours (n = 78) and testing the intestinal environment (n = 14). Through this initiative, we developed a personalised wellness enhancement programme based on collaboration with multiple stakeholders and a framework for using personal data for research and practical purposes. These results provide an approach for promoting voluntary participation and behavioural changes among people, especially under the COVID-19 pandemic, as well as a practical basis for the government, academia, and industry to intervene effectively in raising people’s awareness of health and wellness.

Eukaryotic initiation factor 4A-3 (eIF4A3) is an Asp-Glu-Ala-Asp (DEAD) box-family adenosine triphosphate (ATP)-dependent RNA helicase. Subtypes eIF4A1 and eIF4A2 are required for translation initiation, but eIF4A3 participates in the exon junction complex (EJC) and functions in RNA metabolism including nonsense-mediated RNA decay (NMD). No small molecules for NMD inhibition via selective inhibition of eIF4A3 have been discovered. Here, we identified allosteric eIF4A3 inhibitors from a high-throughput screening campaign. Chemical optimization of the lead compounds based on ATPase activity yielded compound 2, which exhibited noncompetitive inhibition with ATP or RNA and high selectivity for eIF4A3 over other helicases. The optimized compounds suppressed the helicase activity of eIF4A3 in an ATPase-dependent manner. Hydrogen/deuterium exchange mass spectrometry demonstrated that the deuterium-incorporation pattern of compound 2 overlapped with that of an allosteric pan-eIF4A inhibitor, hippuristanol, suggesting that compound 2 binds to an allosteric region on eIF4A3. We examined NMD activity using a luciferase-based cellular reporter system and a quantitative real-time polymerase chain-reaction-based cellular system to monitor levels of endogenous NMD substrates. NMD suppression by the compounds correlated positively with their ATPase-inhibitory activity. In conclusion, we developed a novel eIF4A3 inhibitor that targets the EJC. The optimized chemical probes represent useful tools for understanding the functions of eIF4A3 in RNA homeostasis.

General control nonderepressible 2 (GCN2) is activated by the accumulation of uncharged tRNA in response to amino acid shortage and regulates amino acid starvation response in the cytosol. Here we report the nucleolar localization of GCN2 and the association between GCN2 and small RNA transcripts. Immunofluorescence analysis revealed that GCN2 was constitutively localized to the nucleolus or recruited to the nucleolus by amino acid starvation stress. The nucleolus is the largest structure in the nucleus, where it primarily serves as the site of ribosome and RNA synthesis in addition to acting as a stress sensor through the regulation of p53 function. We found that siRNA-mediated depletion of GCN2 increases small RNA transcripts such as tRNA and 5S rRNA, and induces the p53 pathway activation. Derepression of these transcripts and p53 pathway activation by GCN2 depletion was restored by depletion of B-related factor 1 (BRF1), a primary subunit of RNA polymerase III (pol III) components. These data suggest that the excess amount of small RNA transcripts following GCN2 depletion was responsible for the p53 activation. Our findings reveal a role of GCN2 in the nucleolus that is involved in the expression of small RNA transcripts and serves as alternative stress-sensing machinery for nutrient deficiency. Thus, GCN2 may play pivotal roles in multiple protein translation checkpoints in both the nucleolus and cytosol.

Background The aim of the study was to investigate trends in cancer prognosis by examining the relationship between period of diagnosis and probability of death from cancer in a population-based cohort. Methods Within a cohort of Japanese men and women aged 40–69 years and free of prior diagnosis of cancer and cardiovascular disease at baseline, data from 4403 patients diagnosed with cancer between 1990 and 2006 and followed up until 2012 were analyzed using survival regression models to assess the presence of an effect of the period of diagnosis (before 1998 versus after 1998) on the risk of dying from cancer. Results We noted a significant decrease in risk of dying from cancer among individuals diagnosed after 1998 with lung cancer (hazard ratio [HR] = 0.676 [0.571–0.800]) or colorectal cancer (HR = 0.801 [0.661–0.970]). A decrease in the estimated five-year probability of death from cancer was also noted between the first (before 1998) and the second (after 1998) period of diagnosis for lung and colorectal cancers (e.g., 85.4% vs. 73.3% for lung cancer and 44.6% vs. 37.7% for colorectal cancer, respectively, for stage III in men aged 60 at diagnosis). Conclusions This study presented the first scientific evidence of improvement in prognosis for lung and colorectal cancer patients in a population-based cohort in Japan. Our results suggest that recent advances in cancer treatment could have influenced cancer survival differently among lung, colorectal and gastric cancers.

Nowadays, decreasing birthrate and aging population become to be the serious social problem in Japan. Therefore, needs of assistive devices are more and more increasing. When people use such assistive devices, there is a risk that devices cost user excessive burden. So analysis and evaluation of motions and load of person who uses assistive devices are necessary. In this paper, a model to analyze the effect of external force applied to walking person by fall prevention robot is proposed. A way of suppressing human's lower limbs load in fall prevention assist is discussed through experiments.

Scholarly attention to pharmaceutical companies' ability to sustain research and development (R&D) productivity has increased as they increasingly handle business challenges. Furthermore, the deterioration of R&D productivity has long been considered a major cause of mergers and acquisitions (M&As). This study attempts to investigate quantitatively the possible causes of the deterioration and the relationship between the deterioration and M&As by examining the Japanese pharmaceutical industry. Japan from 1980 to 1997 is an ideal case because of the availability of official data, but more importantly the significant changes in its business environment at the time. Using the Malmquist Index and data envelopment analysis, we measured the deterioration of R&D productivity from 1980 to 1997 based on a sample of 15 Japanese companies. Two lessons can be learned from Japan's case. First, to sustain R&D productivity over the long term, companies should use licensing activities and focus on the dominant therapeutic franchises. Second, if a company fails significantly to catch up with the benchmark, it is likely to pursue an M&A or seek an alternative way to improve R&D productivity. These findings appear similar to the current situation of the global pharmaceutical industry, although Japan pursued more licensing activities than M&A to improve R&D productivity.

Chromophobe renal cell carcinoma (RCC) accounts for approximately 5% of renal epithelial neoplasms. Multiple and/or bilateral chromophobe RCCs in an individual are generally rare but frequently occur in patients with Birt-Hogg-Dube syndrome (BHDS) and in patients with tuberous sclerosis complex (TSC). The responsible genes in both BHDS and TSC act as tumor suppressors. Therefore, it seems that some genetic backgrounds are required for the generation and progression of multiple chromophobe RCCs. Here, we report a case of multiple and bilateral chromophobe RCCs along with several small-sized capsular angiomyolipomas known as 'capsulomas' in a 39-year-old woman who had neither a particular medical history nor specific gene mutation. There has been no report of sporadic multiple chromophobe RCCs and 'capsulomas' developing in a patient without genetic features, having potential for novel genetic variation.

Sitagliptin, the first of a new class of dipeptidyl peptidase-4 (DPP-4)-inhibitory oral antihyperglycaemic drugs (OHDs), was introduced in Japan in December 2009. In April 2010 a safety alert was issued regarding the risk of serious hypoglycaemic events, and prescribers were recommended to reduce the dose of sulfonylurea (i.e. glimepiride, glibenclamide [glyburide] or gliclazide) in patients receiving a combination of sulfonylurea and sitagliptin. A propensity score-matched cohort study was performed using Japanese pharmacy prescription receipt data for OHDs in order to confirm reported changes in OHD prescription behaviour for patients receiving sitagliptin before and after the safety alert. Prescription data from about 6,500 medical institutions throughout Japan during December 2009 to 31 December 2010 were randomly collected from 300 pharmacies, covering 82,064 patients with 629,955 prescriptions for OHDs. Patients who had received a sulfonylurea and sitagliptin (1,788 patients/3,576 prescriptions) before the safety alert were designated as the DPP-4 group. Patients who had received a sulfonylurea but not sitagliptin (30,963 patients/61,926 prescriptions) before the alert were designated as the non-DPP-4 group. Propensity score matching was employed to match baseline characteristics, such as age, sex, type of OHD, metformin use, type of prescribers period for measuring baseline period and type of prescribers’ institutions, for 1,783 patients from each group. In the matched cohort, logistic regression analysis was conducted to compare prescription trends before and after the alert. The primary outcome measure of this study was dose of glimepiride, glibenclamide or gliclazide prescribed for DPP-4 and non-DPP-4 patients. In the propensity score-matched cohort, the proportion of glimepiride dose >2 mg of DPP-4 patients was reduced from 45.8 % in Period 1 (before the alert) to 37.5 % in Period 2 (after the alert) (odds ratio [OR] 0.71; 95 % CI 0.579–0.870), whereas in the case of non-DPP-4 patients the proportion was changed from 28.9 % to 29.5 % in the matched cohort (OR 1.03; 95 % CI 0.868–1.215). The mean prescribed glimepiride dose in DPP-4 patients was also reduced from 2.79 ± 1.81 mg in Period 1 (before the alert) to 2.38 ± 1.71 mg in Period 2 (after the alert) [p

Summary What is known and Objective: The increasing cost of drug research and development and the decreasing number of new drugs being launched are serious issues for pharmaceutical companies. Biomarkers for predicting drug effectiveness are regarded as useful tools for combating these trends. However, the extent to which these biomarkers actually help in improving drug development is unclear. Here, we investigated the efficiency of biomarker usage in oncology drug development by focusing on stratification markers. Methods: Anti-tumour agents for which clinical studies were initiated between 1998 and 2009 were identified using commercially available data sources, and clinical trials registered in ClinicalTrials.gov were examined to identify the use of stratification marker. Phase transition probability for each clinical phase was calculated and analysed along with various other factors that may affect the efficiency of the development process. Results and Discussion: Of 908 anti-tumour agents identified, 121 (13·3%) utilized stratification markers in their clinical studies. Phase I, II and III transition probabilities for all agents were 76·4%, 50·8% and 58·5%, respectively. Corresponding Phase I, II and III transition probabilities of agents developed with stratification markers of 90·4%, 69·0% and 85·0%, respectively, were significantly higher than those for agents without stratification markers. Orphan designation positively affected phase transition probabilities of agents without stratification markers in all phases, while it did not affect transition probabilities of agents with stratification markers, except for Phase II. This shows that stratification markers help improve the probability of success in the development of agents without orphan designation. What is new and Conclusion: Stratification markers contribute to improving the efficiency of development of anti-cancer drugs. The majority of non-orphan drugs are still being developed without stratification markers. Finding reliable stratification markers for all drugs should improve the success rates in drug development.

Objective There is little evidence concerning risk factors for sudden cardiac death (SCD) among Asians. Patients and methods A prospective, nested, case-control study of Japanese patients aged between 30 and 84 years was undertaken using data collected from 26 870 participants in cardiovascular risk surveys conducted in four communities between 1975 and 2005. The incidence of SCD was ascertained by systematic surveillance, with 239 cases of SCD identified over this period. For each case of SCD, three control patients were selected, matched by age, sex, examination year, follow-up time, and community. Results Hypertension, diabetes mellitus, smoking, major ST-T abnormalities, left high amplitude R waves, and increased heart rate (≥77 beat/min) were all independently associated with a 1.5-3.2-fold increase in SCD risk, whereas no associations were observed for body mass index and hypercholesterolemia. The population-attributable fraction [95% confidence interval (CI)] was 23.0% (2.9-39.0) for hypertension, 15.3% (3.8-25.5) for current smoking, 14.5% (8.0-20.5) for major ST-T abnormalities, and 8.1% (2.2-13.7) for diabetes mellitus. The number of SCD risk factors (hypertension, diabetes, smoking, and ECG abnormalities) was positively associated with increased SCD risk. The odds ratio for increased SCD risk with three or more risk factors versus zero risk factors was 5.76 (95% CI 3.20-10.39). Conclusions Among the Japanese population, hypertension, smoking, major ST-T abnormalities, left high amplitude R waves, and diabetes mellitus were associated with an increased incidence of SCD, whereas there were no associations of body mass index or hypercholesterolemia with SCD incidence.

Using a database combining survey responses and archival data on 32 venture capital (VC) firms, the authors test whether professional compensation, strategy, and institutional environment affect entrepreneurial financing practices, such as contractual term choice and deal source choice. The results suggest that contract design fundamentally depends on the VC manager’s compensation system (particularly, carried interest compensation), the investor’s leadership in investment syndications, and the institutional environment. VC firms significantly tend to require control rights and liquidation rights in country’s with poor investor protection. The results also show that selection of deal sources depends on co-investment options, investment stage preference, leadership in investment syndications, and industry expertise.

Japanese government has started to implement several actions to promote the use of generics; however, the generic penetration rate in value advanced rather slowly from 15.4% in March 2006 to 18.7% in December 2009. Japanese pharmaceutical market has at least two unique facts which differentiate from other markets: timing of generics launch and pricing. These unique features may require a new entrant to consider the different marketing strategy. In this article, we observed that the penetration rate varies among therapeutic categories and the penetration curve for vasodilators, miscellaneous antineoplastics, and synthetic antibacterials is similar. Particularly, these categories had a launch of major original drugs within the examined period and the logistic regression for each category indicated that the reflection point of the curve was 21, 33, and 39 days after the generics launch, accordingly. As previous research suggests that the estimated sales revenue of the original drug is considered as a key determinant for generic drug penetration even in Japan. Japanese generics market will likely grow with a support of government generic promotion policy and the upcoming patent expiry of blockbusters. It is important to consider the entry strategy by therapeutic category and plan the early market penetration strategy.

Summary What is known and Objective: Concern about the drug lag, the delay in marketing approval between one country and another, for anticancer drugs has increased in Japan. Although a number of studies have investigated the drug lag, none has investigated it in relation to the transition of anticancer therapy from traditional cytotoxic drugs to molecularly targeted agents. Our aim was to investigate current trend in oncology drug lag between the US and Japan and identify oncology drugs approved in only one of the two countries. Methods: Publicly and commercially available data sources were used to identify drugs approved in the US and Japan as of 31 December 2010 and the data used to calculate the drug lag for individual drugs. Results and Discussion: Fifty-one drugs were approved in both the US and Japan, whereas 34 and 19 drugs were approved only in the US or Japan, respectively. Of the 19 drugs approved only in Japan, 12 had not been subject to development for a cancer indication in the US, and all were approved before 1996 in Japan. Of the 34 drugs approved only in the US, 20 had not been subject to development in Japan, and none was in the top 25 by annual US anticancer drug-class sales. For drugs approved in both countries, the mean approval lag of the molecularly targeted drugs (MTDs) was significantly shorter than that of the non-molecularly targeted drugs (non-MTDs) (3·3 vs. 5·4 years). Further, mean R&D time of the MTDs was significantly shorter than that of non-MTDs (10·0 vs. 13·7 years). The price of MTDs had increased on average by 6·6% annually in the US, whereas it had decreased on average by 4·3% biyearly in Japan. What is new and Conclusion: The emergence of new molecularly targeted agents has contributed to reducing the approval lag, most likely due to improvements in R&D strategy.

This study examines 32 independent and corporate venture capital firms investing in biotechnology and medical device companies. When investing funds in entrepreneurial companies, life science venture capitalists prefer direct involvement over involvement through a third-party fund. Early stage and seed stage are the most and least popular stages, respectively, at which venture capitalists prefer to invest. In our sample, 80% of all firms almost always or frequently use carried interest in portfolio businesses and about 20% never use it. The preferences of the venture capital firms that award low- and high-powered incentives in the sourcing of deal flows and designing contracts are significantly different. The incentive system of venture capital firms is shown to play an important role in promoting an active investment behavior.

PurposeThe purpose of this study is to provide a new perspective for effective management of the merger and acquisition (M&A) process by making use of a case study of a merger in the Japanese pharmaceutical industry. As corporate governance and culture are notably employee‐oriented in Japanese firms, the paper seeks to explore whether M&As in the industry may follow different paths from those in Western companies and lead to competitive advantage.Design/methodology/approachThe paper examines the case of a merger of a Japanese pharmaceutical company, Astellas Pharma, which was formed as a result of one of the largest M&As in Japanese M&A history. The case is analyzed in line with previous theories on M&A process management.FindingsThe case was characterized by a strong momentum created by a core merger team consisting of a few members of management and workforce representatives, and the merger process was facilitated by both top‐down leadership and ground‐level support from the workforce. While broad engagement and non‐rapid rationalization of the workforce can slow the execution of the merger process and delay efficiency savings, it can also consistently drive the merger process and place the merged firm on a solid foundation with strong commitment from all levels.Originality/valueThis study suggests a new mode of the M&A process by focusing on a particular merger case of a Japanese pharmaceutical company. The mechanisms observed in the case should be applied with appropriate modifications to the M&As of Western companies.

Summary What is known and objective: A fundamental management imperative of pharmaceutical companies is to contain surging costs of developing and launching drugs globally. Clinical studies are a research and development (R&D) cost driver. The objective of this study was to develop a productivity breakdown model, or a key performance indicator (KPI) tree, for an entire clinical study and to use it to compare a global clinical study with a similar Japanese study. We, thereby, hope to identify means of improving study productivity. Methods: We developed the new clinical study productivity breakdown model, covering operational aspects and cost factors. Elements for improving clinical study productivity were assessed from a management viewpoint by comparing empirical tracking data from a global clinical study with a Japanese study with similar protocols. Results: The following unique and material differences, beyond simple international difference in cost of living, that could affect the efficiency of future clinical trials were identified: (i) more frequent site visits in the Japanese study, (ii) head counts at the Japanese study sites more than double those of the global study and (iii) a shorter enrolment time window of about a third that of the global study at the Japanese study sites. What is new and conclusion: We identified major differences in the performance of the two studies. These findings demonstrate the potential of the KPI tree for improving clinical study productivity. Trade-offs, such as those between reduction in headcount at study sites and expansion of the enrolment time window, must be considered carefully.

製薬業界は，大型製品の特許満了，研究開発の遅れ，新規上市品目数の減少，後発医薬品の市場浸透，薬価制度の変更と，近年，急速に事業環境が大きく変化しており，新たなビジネスモデルの確立が必要となってきている。これらの問題に対しての打開策として，海外大手製薬企業は，他社との経営統合，新興諸国市場への進出，後発医薬品事業の展開など，新しいビジネスモデルの構築を行っており，一方，自社開発製品の海外展開により成長してきた国内大手製薬企業は，海外企業の買収等を行うことで，打開策を模索している。国内では，海外に比べ遅れていた後発医薬品の使用促進が国の主導のもと行われており，国内事業に特化している新薬開発型製薬企業にとっては，製品の特許満了後の後発医薬品へのシフトによる減収リスクが大きくなってきており，収益を長期収載品に依存するビジネスモデルの変革を余儀なくされている。このように，事業環境の変化に対応すべく，近年，製薬企業が取っている動きの大きなポイントとなるものは，1）医薬品兼業企業による経営統合，2）新興諸国への取組み強化，3）後発医薬品の取組み強化，4）企業買収による海外展開に分けられる。今後のジネスモデルの確立と成功が待たれる。

Background: Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the Asp-Glu-Ala-Asp (DEAD) box RNA helicase family. There are three subtypes of eIF4A, eIF4A1, 2, and 3. eIF4A1 and eIF4A2 are required for translation initiation as their name suggest, however eIF4A3 is functionally distinct and one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD), which is the surveillance mechanism that recognizes mRNAs containing premature termination codons to prevent the accumulation of truncated proteins. It is known that eIF4A3 is an ATP-dependent RNA clamp that can serve as a nucleation center to recruit other EJC components, and siRNA-mediated knockdown of eIF4A3 leads to a defect in NMD. In order to investigate the functions of eIF4A3 further and evaluate the therapeutic potential, we conducted a search for molecular probes of eIF4A3. Methods: Using an RNA dependent ATPase assay as a guide, intensive structure activity relationship (SAR) study and chemical optimization of high-throughput screening hit were conducted. Thereafter, NMD inhibitory activity, selectivity, helicase inhibitory activity, and physicochemical properties of optimized compounds were confirmed. Additionally, direct binding of inhibitors to eIF4A3 and their binding mode were analyzed using biophysical methods. Results: Optimized compounds showed high selectivity for eIF4A3 and exhibited significant NMD inhibitory activity in HEK293T cells. The drastic difference in eIF4A3 inhibitory activity between eutomers and distomer revealed the importance of stereochemistry at the 3-position of the piperazine ring for eIF4A3 inhibition. The lack of significant NMD inhibition by distomers suggests that NMD inhibition of eutomers is mediated by eIF4A3 inhibition. A surface plasmon resonance (SPR) biosensing assay and hydrogen/deuterium exchange mass spectrometry (HDX MS) provided strong evidence for the direct binding of the inhibitors to eIF4A3 and implications for their binding site. Conclusion: We discovered the first selective eIF4A3 inhibitors exhibiting cellular NMD inhibitory activity. These novel inhibitors not only represent novel molecular probes for investigation of RNA biology but also could be promising lead compounds for drug discovery research. References: [1] Ito, M.; Tanaka, T.; Cary, D. R.; Iwatani-Y, M.; Kamada, Y.; Kawamoto, T.; Aparicio, S.; Nakanishi, A.; Imaeda, Y. Discovery of novel 1,4-diacylpiperazines as selective and cell-active eIF4A3 inhibitors. J. Med. Chem. 2017, 60, 3335-3351. [2] Iwatani-Y, M.; Ito, M.; Ishibashi, Y.; Oki, H.; Tanaka, T.; Morishita, D.; Ito, T.; Kimura, H.; Imaeda, Y.; Aparicio, S.; Nakanishi, A.; Kawamoto, T., Discovery and characterization of a eukaryotic initiation factor 4A-3-selective inhibitor that suppresses nonsense-mediated mRNA decay. ACS Chem. Biol. 2017, 12, 1760-1768. Citation Format: Masahiro Ito, Misa Iwatani-Yoshihara, Toshio Tanaka, Douglas R. Cary, Yusuke Kamada, Yoshihiro Ishibashi, Hideyuki Oki, Satoshi Sogabe, Shoichi Nakao, Daisuke Morishita, Takashi Ito, Hiromichi Kimura, Tomohiro Kawamoto, Samuel Aparicio, Atsushi Nakanishi, Yasuhiro Imaeda. Discovery of the first selective and cell-active eIF4A3 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2802.

SummaryThe Hedgehog (Hh) pathway plays critical roles in normal development and in tumorigenesis. We generated Gli-luciferase transgenic mice to evaluate the Smo inhibitor, HhAntag, by whole animal functional imaging. HhAntag rapidly reduced systemic luciferase activity in 10- to 14-day-old mice following oral dosing. Although pathway activity was restored 2 days after drug removal, brief inhibition caused permanent defects in bone growth. HhAntag inhibited proliferation and promoted differentiation of chondrocytes, leading to dramatic expansion of the hypertrophic zone. After drug removal, osteoblasts invaded the cartilage plate, mineralization occurred, and there was premature fusion of the growth plate resulting in permanent disruption of bone epiphyses.

The pharmaceutical industry has experienced intermittent waves of mergers and acquisitions (M&As) since the 1980s and recently appeared to be in yet another wave. Previous studies indicated rather negative impacts of consolidation on research and development, suggesting that they do not necessarily lead to long-term reinforcement of research capabilities, although they may enrich the drug pipeline in the short term. However, recent studies have implied a positive side in terms of knowledge-base transfer. Further micro-organizational studies suggested that scientists learned new knowledge and approaches from partner scientists and improved their performance and innovation. These findings imply that measures for the scientist-level integration after M&As would reinforce fundamental research capabilities in the long term.

本研究では，業界再編が著しい本邦の医薬品産業に着目し，企業の合併・買収（M&A）が医薬研究開発（R&D）活動のイノベーション創出に与える影響を評価し，その企業経営における意義を考察した。手法としては，医薬R&D活動のパフォーマンスを評価のうえ，アステラス製薬の合併事例をケーススタディーに採用し，同社の合併成立後２年時点における合併後マネジメントの発現効果とその背後のメカニズムを，R&D規模，研究効率及びライセンス効率の各指標について調査した。調査の結果，合併後の相互補完による技術機会の拡大がR&D規模に確認され，技術機会の拡大による効率向上がR&D効率及びライセンス効率に有意に認められた。加えて，この統合効果が，同社の掲げる企業ビジョンや合併前に立案された戦略と良く一致していることも確認された。先行研究に基づく通説では，製薬企業のM&Aには開発パイプラインの強化など短期的な効果が認められる一方，永続的な生産性改善には必ずしも寄与しないとされる。本研究は，規模の経済のみならず範囲の経済の視点からの医薬R&D活動の経過観察が，製薬企業の合併後マネジメントの評価に必要であることを示唆するものである。

The sonic hedgehog (Shh) pathway is activated in ∼30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes. [Cancer Res 2007;67(8):3871–7]

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment is associated with major long-term side effects; therefore, new nontoxic therapies, targeting specific molecular defects in this cancer, need to be developed. We use a mouse model of medulloblastoma to show that inhibition of the Sonic Hedgehog (Shh) pathway provides a novel therapy for medulloblastoma. A small molecule inhibitor of the Shh pathway, HhAntag, blocked the function of Smoothened in mice with medulloblastoma. This resulted in suppression of several genes highly expressed in medulloblastoma, inhibition of cell proliferation, increase in cell death and, at the highest dose, complete eradication of tumors. Long-term treatment with HhAntag prolonged medulloblastoma-free survival. These findings support the development of Shh antagonists for the treatment of medulloblastoma.

The Sp family is a family of transcription factors that bind to cis-elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC-rich binding site (GGGCGG) in DNA and C-terminal zinc finger motifs in the proteins. In this study, we characterized the GC-rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis-element (nucleotides -161 to -147) was essential for the expression of the mouse gene for Dnmt1. DNA-binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis-element in this region in a dose-dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3.

Endothelial Fas ligand (FasL) contributes to the "immune privilege" of tissues such as testis and eye, in which apoptosis is induced in infiltrating Fas-positive activated T cells and results in the inhibition of leukocyte extravasation. In this study, we examined the role of endothelial FasL in controlling cancer cell transmigration using rat lung endothelial (RLE) cell line bearing a doxycycline-inducible hepatocyte nuclear factor (HNF)-4alpha expression system. We showed that a detectable level of FasL was expressed in RLE cells and that this expression was markedly up-regulated and well correlated to the degree of HNF-4alpha expression in a time-dependent manner. When various cancer cells were overlaid on an RLE monolayer sheet, we examined the ability of endothelial FasL to induce massive apoptosis in Fas-expressing cancer cells and found a causal link to inhibition of the transmigration. Finally, we showed that FasL was expressed in capillaries of the rat brain by immunohistochemical staining, suggesting that FasL serves its functions not only in vitro, but also in vivo. These results raise the possibility that HNF-4alpha is involved in regulating cancer cell transmigration by modulating the Fas-FasL system.

We previously analysed the plasma membrane proteins of rat placenta and prepared a database of 150 plasma membrane proteins, expressed in a stage-specific manner, utilizing two-dimensional gel electrophoresis (2D/E) [Mol. Cell. Endocrinol. 115(1995)149]. In this study, we focused on the proteins, tentatively named psL-I (MW 36.2 kDa, pI 5.3) and psL-II (35.9 kDa, 5.3), which were expressed mainly in late pregnancy. Close to psL-I and psL-II on 2D/E gels, we also recognized more abundant proteins [psC-I (36.2 kDa, 5.4) and psC-II (35.9 kDa, 5.4), respectively] arranged side by side with the same MW but different pI. Expression of psL-I and psL-II was detected only in junctional zone of placenta, whereas psC-I and psC-II were expressed in both labyrinth and junctional zones. In addition, psL-I and psL-II began to increase on day 16 of pregnancy and peaked at term, whereas expression of psC-I and psC-II was relatively constant. The analysis of these four proteins (psL-I, psL-II, psC-I and psC-II) by preparative 2D/E, peptide mapping, amino acid sequence and mass spectrometry (MALDI-TOF-MS) revealed that psC-I was a G protein beta1 subunit, and psC-II was a beta2 subunit, and showed that psL-I and psL-II were molecular modified forms of psC-I and psC-II, respectively. Expression of these G protein beta subunits (psL-I, psL-II, psC-I and psC-II) was also observed in rat choriocarcinoma cells, Rcho-1 cells. Expression of psC-I and psC-II was much higher than those of psL-I and psL-II, and their level was relatively constant regardless of the stage of differentiation in vitro. Interestingly, expression of psL-I and psL-II gradually increased in association with the differentiation. Since the expression of beta1 and beta2 subunit proteins and their mRNAs was constant during the process of differentiation in Rcho-1 cells, the expression of these lower pI forms of G protein subunits (psL-I and psL-II) was thought to be post-translationally regulated. In conclusion, there are modified forms of G protein beta1 and beta2 subunits, in the placenta and Rcho-1 cells, which are expressed in a pregnancy-stage or differentiation stage specific manner.

PAL31 is a nuclear protein expressed by various cell types. In the present study, the expression and function of PAL31 were examined in the cytokine-regulated growth of T and B cell lines. Treatment of the cells with mitogens [ovine PRL, recombinant rat placental lactogen-I (PL-I) and human IL-3] caused a dose-dependent increase in the expression of PAL31 mRNA in the PRL-dependent cell line Nb2, and IL-3 dependent cell line BaF3. A time-course study on synchronized Nb2 cells revealed that the expression of PAL31 is specific to the late G1 and S phases. Immunocytological studies revealed that PAL31 accumulates in the nuclei at the S phase. Furthermore, the antisense oligonucleotide for PAL31 severely inhibited the proliferation of Nb2 cells by inhibiting cells progressing to the S phase. Thus, PAL31 is a nuclear protein associated with cell cycle progression.

Carbonic anhydrase II (CAII) catalyzes the reversible hydration of carbon dioxide and plays key roles in acid base homeostasis in mammals. We found that human CAII gene promoter could be activated in human cells such as HeLa and T47D cells when the CAII promoter-luciferase gene was transfected with v-Src and assayed as a reporter of the promoter activity. Kinase negative mutants of Src, in contrast, showed little activation. The activation was completely suppressed with the introduction of a dominant-negative Ras in T47D cells, while no suppression was observed in HeLa cells. Introduction of various kinds of deletions into the CAII promoter revealed two essential regions responsible for this activation. No activation, however, was observed in activated Fyn-transfected human cells or in v-Src-transfected rodent cells. These findings suggest that Src can modulate the human CAII promoter by exerting its tyrosine kinase activity in certain human cells, and that two types of Src signaling pathways, Ras-dependent and -independent, exist in a cell type dependent manner.

Methylation of genomic DNA is involved in the basic methanism of gene inactivation, chromatin organization, X chromosome inactivation and genomic imprinting. A pattern of DNA methylation is maintained in mitotic cells by DNA (cytosine-5) methytransferase (DNA MTase). The DNA MTase has been shown to be also expressed in postmitotic cells such as neurons. In the present report, as an approch to analyzing mechanisms underlying regulation of DNA MTase expression, we first isolated rat DNA MTase cDNA. The isolated cDNA encoded a protein of 1,622 amino acid residues showing 88.3% and 64.2% of homology with mouse and human DNA MTase, respectively. Northern blot analysis showed that DNA MTase mRNA was highly expressed in placenta during mid- to late- pregnancy. We then analyzed the expression of DNA MTase in Rcho-1 cells, a rat choriocarcinoma-derived cell line, which cease cell division but keep replicating genomic DNA when differentiated in vitro. We found that the expression of DNA MTase protein was decreased in terminally differentiated Rcho-1 cells whereas DNA MTase mRNA was consistently expressed. This result suggested posttranscriptional regulation of DNA MTase activity in Rcho-1 cells. The Rcho-1 cells would be a valuable model for studying the regulation of gene expression and function of DNA MTase in postmitotic, differentiated cells.

The intestinal epithelial barrier restricts the passage of potentially toxic substances into the systemic circulation and is considered to be mostly mediated by tight junctions, though the mechanisms involved in the regulation of intestinal tight junctions are not yet fully understood. In the present study, we examined whether bacterial lipopolysaccharide (LPS) altered the barrier function of tight junction and localization of tight junctional proteins, ZO-1 and 7H6 antigen, in IEC-6 intestinal cells. Administration of LPS to the basolateral surface of IEC-6 cells disrupted the barrier function and caused the disappearance of 7H6 antigen from the cell border, whereas LPS administered to the apical surface altered neither the barrier function nor the localization of 7H6 antigen in IEC-6 cells. On the other hand, the localization of ZO-1 was not influenced by these treatments of LPS. These results suggest that the interaction of LPS with the basolateral surface of intestinal epithelial cells disrupts the barrier function and 7H6 antigen take part in the maintenance of the barrier function in IEC-6 cells. J. Cell. Physiol. 171:284–290, 1997. © 1997 Wiley-Liss, Inc.

Summary What is known and objective Decline in research and development (R&D) productivity and changes in the business environment have led to pharmaceutical company management to strive to improve R&D productivity. This decline is widely considered to be a major cause of industry consolidation and has received increased scholarly attention. This study aims to construct an R&D productivity map to visualize the industry's R&D productivity and to identify similarity in corporate actions with a view to investigate whether there is a relationship between deterioration in R&D productivity and industry consolidation. Methods Research and development productivity is decomposed into two subprocesses to measure productivity: R&D efficiency and R&D effectiveness, and scores were calculated using a two-stage data envelopment analysis (DEA). The map is then constructed by projecting outputs. To identify any relationship between DEA scores and merger and acquisition transactions, a multiple regression model is employed. Results and discussion Data on 21 global pharmaceutical companies, statistical results indicated that companies with lower R&D efficiency scores were more likely to engage in consolidation. Three US companies that were least successful in terms of R&D effectiveness, as measured by our indicators, were either acquired or changed their business model. Conclusion The R&D productivity map is a useful means for visualizing productivity among companies. By grouping companies into four groups, behavioural commonalities can be observed. The R&D productivity map should be useful for monitor the industry's productivity and help to improve it.

Chromophobe renal cell carcinoma (RCC) accounts for approximately 5% of renal epithelial neoplasms. Multiple and/or bilateral chromophobe RCCs in an individual are generally rare but frequently occur in patients with Birt-Hogg-Dubé syndrome (BHDS) and in patients with tuberous sclerosis complex (TSC). The responsible genes in both BHDS and TSC act as tumor suppressors. Therefore, it seems that some genetic backgrounds are required for the generation and progression of multiple chromophobe RCCs. Here, we report a case of multiple and bilateral chromophobe RCCs along with several small-sized capsular angiomyolipomas known as 'capsulomas' in a 39-year-old woman who had neither a particular medical history nor specific gene mutation. There has been no report of sporadic multiple chromophobe RCCs and 'capsulomas' developing in a patient without genetic features, having potential for novel genetic variation.

How natural selection can promote cooperative or altruistic behavior is a fundamental question in biological and social sciences. One of the persuasive mechanisms is "indirect reciprocity," working through reputation: cooperative behavior can prevail because the behavior builds the donor's good reputation and then s/he receives some reciprocal benefits from someone else in the community. However, an important piece missed in the previous studies is that the reputation-building process requires substantial cognitive abilities such as communication skills, potentially causing a loss of biological fitness. Here, by mathematical analyses and individual-based computer simulations, we show that natural selection never favors indirect reciprocal cooperation in the presence of the cost of reputation building, regardless of the cost-to-benefit ratio of cooperation or moral assessment rules (social norms). Our results highlight the importance of considering the cost of high-level cognitive abilities in studies of the evolution of humans' and animals' social behavior.

7H6 antigen is a novel tight junction-associated protein first identified in our laboratory with a monoclonal antibody generated by immunizing mice with a bile canaliculus-rich membrane fraction obtained from adult rat liver. Although the antigen is found preferentially at the tight junction of epithelial cells in various organs in adult mammals, the relationship between 7H6 antigen and other tight junction proteins remains obscure. In the present study we examined the relationship between the immunohistochemical distribution of 7H6 antigen and occludin in chick intestinal epithelial cells during development in vivo, since occludin is the only known membrane protein composing tight junctions which is found thus far exclusively in avian tissues. 7H6 antigen was found as coarse dots along the basolateral cell membrane of intestinal epithelium in the chick embryo, whereas it was found as a fine dashed line along the cell border in the chicken. In contrast, occludin was found linearly together with ZO-1 at the cell border of intestinal epithelia both in the chick embryo and chicken. The possible significance of such different localization is briefly discussed.

Human brucellosis is an imported zoonosis extremely uncommon in Japan. Brucellosis was found in a 38-year-old surgeon who had never been abroad; he developed intrapulmonary infiltrates and pleural thickening indistinguishable from pulmonary tuberculosis. Despite extensive antibiotic therapy for tuberculosis he developed systemic serositis. A culture of resected lung tissues grew CO2-required coccobacilli. Polymerase chain reaction test using a specific primer pair for Brucella abortus revealed that the isolated pathogen is highly homologous to B. abortus. We emphasize that undetermined cases of human brucellosis may be present in Japan.