DNA methylation is one of the major epigenetic marks in the mammalian genome to define chromatin higher-order structure, and plays essential roles in various developmental processes. In the mammalian genome, DNA methylation mainly occurs at the 5th position of cytosine bases in a palindromic 5′-CG-3′dinucleotide sequence. Methyl CpG binding domain (MBD) proteins recognize symmetrically methylated CpG sites (5mCG/5mCG) through a conserved MBD, and recruit transcriptional repressors or chromatin modifiers. One of the MBD proteins, MBD4, uniquely contains a C-terminal glycosylation domain together with an N-terminal MBD, and functions as a mismatch DNA repair enzyme specific for T/G or U/G mismatch bases generated by spontaneous deamination of 5-methylcytosine. The base excision activity of MBD4 is also implicated in active DNA demethylation initiated by the conversion of 5-methylcytosine to thymine by deaminases. Unlike other MBD proteins, MBD4 recognizes not only 5mCG/5mCG but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine (5mCG/TG). In addition, our biochemical data demonstrate that MBD also binds to intermediates in DNA demethylation pathways, such as 5-hydroxymethyl-cytosine (hmC), 5-carboxyl-cytosine and 5-hydroxy-uracil. The crystal structures of MBDMBD4 in complex with 5mCG/TG, 5mCG/5mCG or 5mCG/hmCG provide new structural insights into the versatility of base recognition by MBD4. A DNA interface of MBD4 has flexible structural features, in which an extensive hydration water network supports the versatile base specificity of MBD4. The versatile base recognition by MBDMBD4 implies multi-functional roles of MBD4 in the regulation of dynamic DNA methylation patterns.
Dysfunction of ribosome biogenesis is commonly found in cancers. Because the transcription of ribosomal RNA genes (rDNA) is a rate-limiting step in ribosome biogenesis and is elevated in many cancer cells, ribosomal RNA transcription can be a target for cancer therapy. In eukaryotes, ribosomal RNA is transcribed specifically in nucleoli by RNA polymerase I but not by RNA polymerase II. Therefore, ribosomal RNA transcription by RNA polymerase I would have a distinct nature compared to transcription by RNA polymerase II. Genomic DNA with proteins including histones constitutes chromatin. The structure of chromatin has plasticity and is regulated by chemical modifications of chromatin's components. We had reported that histone demethylase KDM2A reduced ribosomal RNA transcription in response to starvation. In this symposium, we reported our recent results showing the mechanism by which KDM2A was recruited to rDNA chromatin. We found that KDM2A bound to a rDNA promoter with unmethylated CpG dinucleotides via KDM2A CxxC-zinc finger motif. This binding was required for KDM2A to demethylate histone in the rDNA promoter and reduce rDNA transcription resulting from starvation. Further, this binding was detected before starvation, independent of the demethylase activity. We also found that the histone demethylation by KDM2A in response to starvation was detected only in the rDNA promoter, but not in a gene promoter transcribed by Pol II, the P2RX4 promoter. These results suggest that it is important to consider genome regions and cell conditions when developing epigenetic drugs.
Fluorescence imaging using synthetic probes is becoming increasingly popular as a chemistry-based technique for the analysis of biomolecules. Real-time visual information on various biological molecules can be obtained by designing probes with high flexibility. The focus of our research is to design fluorogenic probes which are non-fluorescent in their initial intact form, but exhibit enhanced fluorescence intensity upon reactions with target biomolecules. The fluorescence switch of these probes allows the detection of the function and the localization of biomolecules with a high signal-to-noise ratio. Thus far, we have succeeded in designing and synthesizing fluorogenic probes that visualize molecules involved in epigenetics. Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression. A significant amount of epigenetic information can be obtained by the detection of enzyme activity. However, the existing methods require complicated multistep procedures. To overcome this limitation, we developed fluorogenic probes for the detection of HDAC activity in a one-step procedure. In this review, the details of the strategy for probe design and the detection method have been described.
The aging process is largely influenced by dietary factors. For example, caloric restriction can slow age-related functional deterioration and the onset or progression of age-related diseases, as well as prolong mean and maximum life span in laboratory animals. However, the dietary factors that affect the aging process comprise not only calories, but also various nutrients, such as proteins, carbohydrates, fats, and vitamins. Phytochemicals, which are found in plants, are non-nutritive, yet many phytochemicals are known to act as antioxidants and prevent diseases associated with free radical production. Furthermore, certain phytochemicals can help prevent or reduce the risk of cancer, inflammation, and cardiovascular disease by alteration of several signal transduction pathways in cells. Therefore, much focus is being placed on the effects of dietary phytochemicals on aging and stress response. This paper reviews recent advances in the study of two major dietary phytochemicals, resveratrol and curcumin, on aging and stress response.
The high prevalence of dementia in aged individuals suggests that aging is the most important risk factor and that senescence further enhances dementia. We have searched for dietary factors that prevent brain senescence using a mouse model of age-related neurodegeneration (SAMP10). This mouse line is suitable for studying brain senescence because brain atrophy and cognitive dysfunction are observed with aging, similar to humans. The production of reactive oxygen species and oxidative damage are high in the brains of aged SAMP10. We found that green tea catechin and β-cryptoxanthin in Japanese mandarin oranges prevented brain atrophy and cognitive dysfunction. In addition, psychosocially chronically stressed mice exhibited a shortened life span and accelerated cognitive dysfunction. These deficiencies were prevented by the ingestion of theanine, an amino acid in tea, under stressed conditions. While a number of factors affect brain senescence, our results suggest that non-nutritive food components such as catechin, β-cryptoxanthin and theanine may be useful for preventing brain senescence.
We previously reported that zerumbone, a sesquiterpene found in Zingiber zerumbet SMITH, showed notable cancer preventive effects in various organs of experimental rodents. This agent up-regulated nuclear factor-E2-related factor (Nrf2)-dependent expressions of anti-oxidative and xenobiotics-metabolizing enzymes, leading to an increased self-defense capacity. On the other hand, zerumbone markedly suppressed the expression of cyclooxygenase-2, an inducible pro-inflammatory enzyme, by disrupting mRNA stabilizing processes. Binding experiments using a biotin derivative of zerumbone demonstrated that Keap1, an Nrf2 repressive protein, is one of its major binding proteins that promotes their dissociation for inducing Nrf2 transactivation. We then generated a specific antibody against zerumbone-modified proteins and found that zerumbone modified numerous cellular proteins in a non-specific manner, with global distribution of the modified proteins seen not only in cytoplasm but also the nucleus. Based on those observations, zerumbone was speculated to cause proteo-stress, a notion supported by previous findings that it increased the C-terminus of Hsc70 interacting protein-dependent protein ubiquitination and also promoted aggresome formation. Interestingly, zerumbone counteracted proteo-stress and heat stress via up-regulation of the protein quality control systems (PQCs), e.g., heat shock proteins (HSPs), ubiquitin-proteasome, and autophagy. Meanwhile, several phytochemicals, including ursolic acid and curcumin, were identified as marked HSP70 inducers, whereas most nutrients tested were scarcely active. Recent studies have revealed that PQCs play important roles in the prevention of many lifestyle related diseases, such as cancer, thus non-specific binding of phytochemicals to cellular proteins may be a novel and unique mechanism underlying their physiological activities.
One of the factors that is believed to contribute to Japanese longevity is their dietary life, which is unique and distinct from Westerners. However, there has been no study that examined whether Japanese food in itself is effective for health maintenance. Therefore, we investigated in rats whether Japanese food is beneficial to health maintenance compared with American food. As a result, we revealed that modern Japanese food is useful for health maintenance compared with modern American food. We subsequently investigated the health benefits of Japanese food from different eras. The menus of Japanese foods from 4 different years, 2005, 1990, 1975 and 1960 were prepared, cooked, and powderized. Each of the Japanese foods was provided to mice. We found that the Japanese food from 1975 exhibited health benefits with respect to the stages of growth, adolescence, maturity and old age. Furthermore, we focused on fish oil, which is one of the beneficial ingredients of Japanese food, and investigated the effect of its long-term intake on lifespan in mice. Surprisingly, long-term intake of large amounts of fish oil shortened the lifespan of these mice. By contrast, intake of small amounts of fish oil with antioxidants extended the lifespan. Moreover, while intake of large amounts of fish oil also shortened the lifespan of Caenorhabditis elegans (C. elegans), intake of small amounts of fish oil extended the lifespan. Thus, it was suggested that even when foods have been reported to have health benefits, ingestion of large amounts of individual foods is undesirable for health maintenance.
Antioxidant micronutrients, such as vitamins and carotenoids, exist in abundance in fruits and vegetables and have been known to contribute to the body's defense against reactive oxygen species. Numerous recent epidemiologic studies have demonstrated that a high dietary consumption of fruit and vegetables rich in carotenoids or with high serum carotenoid concentrations results in lower risks of certain cancers, diabetes, and cardiovascular disease. These epidemiologic studies have suggested that antioxidant carotenoids may have a protective effect against several lifestyle-related diseases. β-Cryptoxanthin is a carotenoid pigment found in Japanese mandarin (Citrus unshiu MARC.) fruit, which is mainly produced in Japan. Our nutritional epidemiologic survey, the Mikkabi Study, utilized data derived from health examinations of inhabitants performed in the town of Mikkabi in Shizuoka, Japan. In this survey, we measured serum β-cryptoxanthin as a specific biomarker to estimate the consumption of Japanese mandarin fruit. From the cross-sectional analyses from the Mikkabi Study, we found inverse associations of serum β-cryptoxanthin with the risks for atherosclerosis, insulin resistance, liver dysfunction, metabolic syndrome, low bone mineral density, and oxidative stress. In this review, recent epidemiologic studies about the associations between serum β-cryptoxanthin with the risk for several lifestyle-related diseases were reviewed.
In 2013, a program to be implemented by the Japan Accreditation Board for Pharmaceutical Education, Third-party Pharmaceutical Education Evaluation, was initiated. The purposes of this program are: 1) to ensure the quality of education programs; 2) to promote their improvement; and 3) to provide support to actively obtain public understanding and cooperation. In the next 7 years, all pharmaceutical departments and universities will undergo this third-party evaluation. The evaluation standards specify the necessity of establishing indices to evaluate goal achievement levels and to ensure appropriate evaluation based on standards in a number of very important domains of pharmaceutical education. They also emphasize that it is necessary to similarly evaluate overall learning achievement levels in undergraduate degree accreditation. On the other hand, the revision of pharmaceutical education models and core curricula was also proposed and considered in 2013, and preparations for the adoption of revised curricula are currently proceeding. The revised models and core curricula are mainly characterized by setting learning goals within a learning-achievement-based education approach, taking into consideration the 10 qualities to be achieved before graduating. In line with this, in order to nurture pharmacists who appropriately meet social needs by enhancing the quality of their education and training, it is crucial for universities to establish methods to appropriately evaluate learning goal achievement levels as a basis for the realization of these qualities. In this article, appropriate pharmaceutical education curricula, particularly evaluation methods, will be discussed from the above-mentioned perspectives, while explaining the related third-party evaluation standards and their viewpoints.
Education in university is commonly based on active learning by the students themselves. Lectures, exercises, and training are major learning strategies in a university. Self-study is one of the methods of active learning and is considered to be a major part of the classes when calculating the credits. When the six-year pharmaceutical education program was started, degree of attitude education was increased dramatically. New learning strategies considered to be suitable for attitude education, such as small group discussion (SGD), World Café, team-based learning (TBL), and problem-based learning (PBL)-Tutorials, were introduced in the classes, and the students were very much attracted by these methods. Not only the tactics but also the skills and abilities of teachers are greatly influenced by using such strategies to realize efficient education. Therefore, the most important point becomes faculty development. The degree of learning and the satisfaction of the students are not always mutually related. The evaluation of learning strategies has become difficult because of ambiguous criteria. Whereas an integrated educational program of drug therapies stimulates the motivation for learning of the students, a well-designed program may ruin the delight in learning of students.
To deal with declining levels of academic ability and motivation among students (a situation attributable to fewer high school graduates, a greater number of universities, and the diversification of entrance examination methods), one must comprehend the conditions of faculties collectively, and take appropriate measures. Using the results of examinations carried out in each grade as indices, we examined levels of academic ability and established various support programs based on the results. Basic chemistry, biology, and physics courses were designed to help first-year students acquire essential academic skills. For second, third, and fourth-year students, two types of support programs were implemented: supplementary instruction to help students improve their understanding of basic topics in pharmaceutical sciences, and an e-learning system to promote self-study, requiring minimal assistance from teachers. Although educational benefits were observed in many students, the number of learners whose understanding failed to improve as a result of the support programs continued to increase. Consequently, The Support Section for Pharmaceutical Education opened in October 2011 to address these concerns. The support section functions mainly to provide individual assistance to students who lack strong academic abilities, and provides teachers with information useful for educational reform. Here, we describe the educational support provided by our faculty and its effectiveness.
A revised core curriculum model for pharmaceutical education, developed on the basis of the principles of outcome-based education, will be introduced in 2015. Inevitably, appropriate assessments of students' academic achievements will be required. Although evaluations of the cognitive domain can be carried out by paper tests, evaluation methods for the attitude domain and problem-solving abilities need to be established. From the viewpoint of quality assurance for graduates, pharmaceutical education reforms have become vital to evaluation as well as learning strategies. To evaluate student academic achievements on problem-solving abilities, authentic assessment is required. Authentic assessment is the evaluation that mimics the context tried in work and life. Specifically, direct evaluation of performances, demonstration or the learners' own work with integrated variety knowledge and skills, is required. To clarify the process of graduate research, we obtained qualitative data through focus group interviews with six teachers and analyzed the data using the modified grounded theory approach. Based on the results, we clarify the performance students should show in graduate research and create a rubric for evaluation of performance in graduate research.
Interprofessional work (IPW) is increasingly important in various setti^ngs including primary care, in which the role of pharmacists is particularly important. Many studies have shown that in cases of hypertension, diabetes, dyslipidemia, and metabolic syndrome, physician-pharmacist collaboration can improve medication adherence and help to identify drug-related problems. Some surveys and qualitative studies revealed barriers and key factors for effective physician-pharmacist collaboration, including trustworthiness and role clarification. In Japan, some cases of good collaborative work between pharmacists and physicians in hospitals and primary care settings have been reported. Still, community pharmacists in particular have difficulties collaborating with primary care doctors because they have insufficient medical information about patients, they feel hesitant about contacting physicians, and they usually communicate by phone or fax rather than face to face. Essential competencies for good interprofessional collaboration have been proposed by the Canadian Interprofessional Health Collaborative (CIHC): interprofessional communication; patient/client/family/community-centered care; role clarification; team functioning; collaborative leadership; and interprofessional conflict resolution. Our interprofessional education (IPE) team regularly offers educational programs to help health professionals learn interprofessional collaboration skills. We expect many pharmacists to learn those skills and actively to facilitate interprofessional collaboration.
The concept of interprofessional work (IPW) is becoming increasingly important recently and the role of nurses in IPW seems critical. In Japan, the problem of burnout and turnover of nurses has been recognized, and the solution seems embedded in the scheme of IPW, because it appears to improve their job satisfaction and recognition as health professionals. However, many obstacles lie ahead, such as “tribal conflict” between health professionals including between pharmacists and nurses. Although failure to understand the roles of other professionals or competencies may seem to hamper with the promotion of collaboration, we must realize that even a lack of understanding among nurses exists. The authors believe that the solution is to understand and respect not only other professionals but also colleagues of the same profession.
In Japan, many health professionals do not seem to have adequate understandings of the competencies and specialties of other professionals. The competencies of pharmacists are especially overlooked because their expected roles have been changing rapidly in recent years and may vary depending on context in various settings. Due to this lack of understanding physicians themselves often perform therapeutic drug monitoring (TDM), and pharmacists sometimes suggest unnecessary prescription proposals to physicians without considering their treatment plans. Although “community and hospital pharmacist collaboration” has been emphasized for many years, many pharmacists in both settings still do not understand one another's situation. How can we make pharmacists' competencies appropriately understood and respected by other health professionals? What should we do to promote interprofessional work (IPW) with more incorporation of pharmacists so as to pursue patient/client/family-centered care? The key to success in IPW seems to lie in the answers to these questions.
To work collaboratively in healthcare practice, health professionals should learn not only the competencies of their own specialties but also those of other professions so as to promote effective interprofessional work (IPW), thus optimizing patient/client outcomes. For this reason interprofessional education (IPE) is urgently needed. Since the establishment of Centre for the Advancement of Interprofessional Education (CAIPE) in 1987, many IPE programs have been developed and implemented worldwide. Currently, our Japan Society for Instructional Systems in Healthcare (JSISH) IPE program project team is conducting a study to develop an effective and versatile IPE program according to the framework of instructional design (ID). The main categories of learning goals of our program are intellectual skills and attitudes among Gagne's five categories. Therefore we designed our program to start from the drama (skit or video) of a bad example of IPW for learners to see and discuss the barriers of interprofessional collaboration. The drama of IPW seems to enhance attention and relevance for learners; both of which are components of the ARCS model. We expect every health professional including pharmacists to learn about IPW competencies through participating in our IPE program, enabling us further to pursue the ideal patient/client/family-centered care together.
As I was deeply interested in the effects of drugs on the human body, I chose pharmacology as the subject of special study when I became a 4th year student at Shizuoka College of Pharmacy. I studied abroad as a postdoctoral fellow for two years, from 1978, under the tutelage of Professor Henry I. Yamamura (pharmacology) in the College of Medicine at the University of Arizona, USA. He taught me a variety of valuable skills such as the radioreceptor binding assay, which represented the most advanced technology developed in the US at that time. After returning home, I engaged in clarifying receptor abnormalities in pathological conditions, as well as in drug action mechanisms, by making the best use of this radioreceptor binding assay. In 1989, following the founding of the University of Shizuoka, I was invited by Professor Ryohei Kimura to join the Department of Pharmacokinetics. This switch in discipline provided a good opportunity for me to broaden my perspectives in pharmaceutical sciences. I worked on evaluating drug-receptor binding in vivo as a combined index for pharmacokinetics and pharmacological effect manifestation, with the aim of bridging pharmacology and pharmacokinetics. In fact, by focusing on data from in vivo receptor binding, it became possible to clearly rationalize the important consideration of drug dose-concentration-action relationships, and to study quantitative and kinetic analyses of relationships among pharmacokinetics, receptor binding and pharmacological effects. Based on this concept, I was able to demonstrate the utility of dynamic analyses of drug-receptor binding in drug discovery, drug fostering, and the proper use of pharmacokinetics with regard to many drugs.
Numerous rodent models of depression have been reported, most requiring a long experimental period and significant effort. We explored a new potential mouse model for depression by investigating whether exposure to a 15-day chronic stress paradigm could induce depression-like behavior in ICR mice. Animals in the stress-exposed groups were subjected to 3 h of restraint while immersed in a 28°C water bath daily for 15 consecutive days. Immobility time in the forced swim test was increased in the chronic stress-exposed mice compared with the controls. Serum corticosterone levels were also much higher in the stressed mice than in the control mice. Hippocampal cell survival (BrdU-positive cells) and neurotrophic factor (NGF, TrkA) mRNA levels were significantly decreased in the chronic stress-exposed mice compared with controls. Administration of the anti-depressant drugs clomipramine (20 mg/kg/d) or imipramine (30 mg/kg/d) did not change the immobility time in the forced swim test, but treatment with lithium (100 mg/kg/d) did result in slight improvement. These results suggest that this 15-day chronic stress paradigm can induce depression-like behavior and neurological changes, in a short time and with minimal effort, facilitating the assessment of treatments for depression.