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.
Parkinson's disease (PD) is an age-related neurodegenerative disorder in whose brain massive loss of dopaminergic neurons and formation of Lewy bodies occur in the substantia nigra (SN). L-Dihydroxyphenylamine (L-DOPA) substitution is still considered the gold standard of antiparkinsonian drug therapy. However, there has been little information available on neuroprotective and regenerative therapies. Recently, we have found that pramipexole and talipexole (D2/D3-dopaminergic agonists) inhibit dopaminergic neurotoxin-induced production of reactive oxygen species and apoptotic cell death. In addition, treatment with these drugs induces enhancement of anti-apoptotic Bcl-2 expression and inhibition of α-synuclein aggregation. Interestingly, recent study suggests that pramipexole treatment delays the progression of early PD symptom. On the other hand, we investigated the transplantation strategy for PD by assessing whether double-transplants of mouse embryonic stem (ES) cell-derived neurons in the striatum (ST) and SN, or subthalamic nucleus (STN), induce functional recovery in rat hemi-parkinsonian model. The study indicates that both the involvement of ST as a place of transplantation and the number of ES cell-derived neurons are essential factors for efficacy on PD animal model. Interestingly, an invertebrate planarian can regenerate complete organs, including a well-organized central nervous system (brain), within about 7 days. The regeneration process of the planarian dopaminergic neural network (tiara) may be divided into five steps: 1) anterior blastema formation, 2) brain rudiment formation, 3) brain pattern formation, 4) the formation of dopaminergic tiara, and 5) functional recovery of dopaminergic motor regulation, with several kinds of genes and molecular cascades acting at each step.
The ubiquitin-proteasome system (UPS) plays a major role in selective protein degradation and regulates various cellular events. Approval of bortezomib for the treatment of multiple myeloma validated the proteasome as an anticancer target. In order to find drug candidates targeting the ubiquitin-dependent protein degradation, we paid an attention to inhibitors against three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), which are required for polyubiquitination of proteins and prerequisite to proteasome-mediated protein degradation. We succeeded in isolating various compounds with three distinct inhibitory activities against an E1 enzyme reaction, Ubc13 (E2)-Uev1A interaction, and p53-HDM2 (E3) interaction as well as the proteasome inhibitors. We also isolated new alkaloids, notoamides, from a marine-derived Aspergillus sp. Among them, notoamide B and stephacidin A contain a bicyclo[2.2.2]diazaoctane ring in their structures. We proposed this ring is constructed from notoamide E by the intramolecular Diels-Alder (IMDA) reaction. Recently, the isolation of the antipodes of notoamides from the terrestrial Aspergillus has been reported. We propose that each enantiomer is generated by a distinct face-selective IMDA.
In this review, I report highly emissive pyrene-based fluorophores and highly sensitive fluorescent sensors using pyrene emission switching. Alkynylpyrene derivatives show fruitful photophysical properties, as compared with pyrene itself: longer absorption and emission wavelengths and high fluorescence quantum yields in the presence of O2. Alkynylpyrene-based fluorescent probes allow the highly sensitive detection of biomolecules such as proteins. Additionally, a rotaxane-based fluorophore, consisting of an alkynylpyrene derivative and two cyclodextrins, is highly stable upon irradiation with a UV/vis illuminator. On the other hand, I and my colleagues have developed a variety of fluorescent sensors using pyrene emission switching. One of them is DNA probes in which two pyrenes are connected both at 3′ and 5′ ends of a single-stranded oligonucleotide. Emission switching occurs from excimer to monomer when the probes hybridized with target DNAs. The DNA probes met antibody structures to produce the following fluorescent sensors. The sensors consist of three functional regions, crown ether (or cyclodextrin), DNA, and pyrene as guest-binding, dimerizing, and sensing sites, respectively. These sensors can detect potassium cation, porphyrin, unsaturated fatty acid by pyrene monomer-excimer switching.
Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. It is reported that stomatin regulates the gating of acid-sensing ion channels in mammalian neurons. However, the function of stomatin is not fully understood. In the genomic sequence of the hyperthermophilic archaeon Pyrococcus horikoshii, the putative operon-forming genes PH1511 and PH1510 encode stomatin and its partner protein, respectively. The N-terminal region of PH1510p (1510-N) is a serine protease, and specifically cleaves the C-terminal hydrophobic region of stomatin PH1511p. We have determined the first crystal structure of the core domain of stomatin PH1511p (residues 56-234, designated as PhStoCD). This review focuses on the three-dimensional structure of PhStoCD, and discusses the function of stomatin and its specific protease 1510-N. PhStoCD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhStoCD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows a conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled-coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. The coiled-coil fold observed in the crystal probably contributes to the self-association.
Deposition of insoluble amyloid fibrils in tissues is a common hallmark of a wide range of human diseases referred to as amyloidoses, including Alzheimer's disease, type II diabetes mellitus. The amyloid deposits cause cell dysfunction, death, and subsequently severe impairment in tissues. Elucidation of amyloid formation mechanisms is essential for prevention of the onset and development of amyloidoses. Accumulated experimental evidence demonstrates that membrane lipids enhance the fibril formation of amyloidogenic proteins. Our group demonstrated that amyloid formation by amyloid β-protein (Aβ) was facilitated by gangliosides in lipid raft-like model membranes. Phosphatidylserine and phosphatidylglycerol were also reported to trigger fibril formation by human islet amyloid polypeptide (hIAPP). However, it is not verified whether the proposed lipid-protein interactions can occur on plasma membranes of live cells. The author developed a method for visualizing amyloid fibrils on live cell membranes and investigated the roles of gangliosides and cholesterol in lipid rafts for amyloid formation. Congo red, an amyloid-specific dye, was found to be a promising compound for staining amyloids in live cells. Aβ was accumulated on cholesterol-dependent ganglioside-rich domains in PC12 neuronal cells in a time- and concentration-dependent manner, leading to cell death. Nerve growth factor-induced differentiation of PC12 cells increased both gangliosides and cholesterol and thereby greatly potentiated the accumulation and cytotoxic effect of Aβ. Amyloid formation by hIAPP was also facilitated by gangliosides in lipid rafts. Membrane lipid compositions, in this case, gangliosides in lipid rafts, actually caused striking change in amyloid formation on cell membranes.
Clinical studies show that galantamine, a weak acetylcholine (ACh) esterase inhibitor and allosteric potentiator of nicotinic ACh receptors (nAChRs), improves negative and cognitive symptoms in schizophrenia, while donepezil, a potent ACh esterase inhibitor, does not. We have recently found that galantamine, but not donepezil, reversed isolation rearing-induced deficits of prepulse inhibition (PPI), sensory information-processing deficits, in mice. In addition, we unexpectedly found that the galantamine-induced improvements in PPI deficits were prevented by the muscarinic ACh receptor (mAChR) antagonists scopolamine and telenzepine (preferential for M1 subtype), but not by the nAChR antagonists. Galantamine, like donepezil, increased extracellular ACh levels in the prefrontal cortex. However, donepezil, unlike galantamine, inhibited M1-mAChR-mediated Ca2+ signal in human neuroblastoma SH-SY5Y. These results suggest that galantamine improves isolation rearing-induced PPI deficits via an activation of mAChRs and the difference in the effect on the PPI deficits between galantamine and donepezil is due to that in their action on M1-mAChRs. The possible mechanisms for the beneficial effect of galantamine are discussed in a model of isolation rearing-induced PPI deficits.
A mechanism-based logical approach is a mainstream of current novel drug therapy development in the context of these trends and, therefore, the elucidation of gene function and the molecular level mechanism analysis of diseases at an individual level in mammals are essential in addition to that in cultured cells. In vivo gene transfection techniques are also indispensable for these purposes as well as the evaluation of gene therapy and nucleic acid-based therapy approaches and clinical applications during the process of development of novel drug therapies. Various recombinant virus and synthetic carrier-mediated transfection methods have been reported, however, above all, naked plasmid DNA transfection without virus vectors, synthetic carriers and special physical devices has attracted much attention, because of its advantages including convenience of preparation and handling and lack of toxicity associated with the transfection agents. In this review, I collect the information of these naked plasmid DNA transfection methods involving tissue pressure-mediated transfection from the comprehensive view point including side effects. Additively, the key physiological phenomena affecting transgene expression, especially activation of transcriptional factors, are reviewed. Combined with conventional approach based with biodistribution control, regulation of physiological change in transfected cells will provide spatial- and temporal-controlled transgene expression at various organs, which leads us to elucidate mechanism of diseases and to develop novel drug therapy in near future.
Quinones are compounds that have various characteristics such as a biological electron transporter, an industrial product and a harmful environmental pollutant. Therefore, an effective determination method for quinones is required in many fields. This review describes the development of sensitive and selective determination methods for quinones based on some detection principles and their application to analyses in environmental, pharmaceutical and biological samples. Firstly, a fluorescence method was developed based on fluorogenic derivatization of quinones and applied to environmental analysis. Secondly, a luminol chemiluminescence method was developed based on generation of reactive oxygen species through the redox cycle of quinone and applied to pharmaceutical analysis. Thirdly, a photo-induced chemiluminescence method was developed based on formation of reactive oxygen species and fluorophore or chemiluminescence enhancer by the photoreaction of quinones and applied to biological and environmental analyses.
Biotransformation is the major clearance mechanism of therapeutic agents from the body. Biotransformation is known not only to facilitate the elimination of drugs by changing the molecular structure to more hydrophilic, but also lead to pharmacological inactivation of therapeutic compounds. However, in some cases, the biotransformation of drugs can lead to the generation of pharmacologically active metabolites, responsible for the pharmacological actions. This review provides an update of the kinds of pharmacologically active metabolites and some of their individual pharmacological and pharmacokinetic aspects, and describes their importance as resources for drug discovery and development.
1-Azabicyclo [1.1.0] butane (ABB) bearing the highly strained bicyclic structure, which is synthetically useful for the preparation of 3-substituted azetidines, was obtained by the cyclization of 2,3-dibromopropylamine hydrobromide derived from inexpensive allylamine only with organolithium compounds and lithium amides. When other bases were employed, such as potassium, sodium, and magnesium species, the reaction yielded almost no ABB. It was speculated that a lithium cation played an important role in the cyclization. Thus, we proposed that this reaction proceeded by the consecutive cyclization to aziridines via the SN2 process involving the activation of the C-Br bond based on the intermolecular Br…Li+ coordination, as a result of studies of reaction mechanisms.
As it is an urgent issue to contain increasing healthcare expenditures, unlimited reimbursement of pharmaceuticals continues to be controversial. The objective of this study is to identify acceptable incremental cost effectiveness ratios between new and conventional therapies. Clinical study data for five statin therapies were used to indicate treatment effectiveness and incremental costs were indicated by price premiums at price listing. The incremental cost effectiveness ratios to pravastatin were 0 yen/patient with response, 1,475.1 yen/patient with response, 3,033.3 yen/patient with response, and 3,032.4 yen/patient with response. By conducting further analyses in various pharmaceuticals and categorizing acceptable incremental cost effectiveness ratios based on the disease severity and expected level of improvement in disease condition, drug prices that reflect the value of new pharmaceuticals and that are reasonable to be reimbursed can be suggested.
For the purpose of enhancing the anticancer potency of docetaxel, a novel excipient, cholesterol-PEG-folate (α-(3.beta) cholest-5-en-3-ω-folic acid-poly (oxy-1, 2-ethanediyl)), was synthesized and used for the preparation of liposomes (folate-conjugated PEG-liposomes). The in vitro release properties, in vitro cytotoxicity, in vivo pharmacokinetics and distribution, as well as in vivo potency of the liposomes were evaluated. These liposomes were able to control the release of the loaded drug. Docetaxel-loaded, folate-conjugated PEG-liposomes were more cytotoxic to MCF-7cells than ordinary PEG-liposomes. The pharmacokinetic parameters of folate-conjugated PEG-liposomes were studied in rats. Compared to docetaxel solution, the folate-conjugated PEG-liposomes enhanced the t1/2 of docetaxel by 6.74-fold. The biodistributions of docetaxel in the heart, brain and kidneys decreased when delivered in liposomes. The folate-conjugated PEG-liposomes could significantly enhance tumor accumulation of docetaxel and antitumor activity in tumor-bearing mice (p<0.05). The precent results indicate that these folate-conjugated PEG-liposomes might enhance the potency while preventing the side effects of docetaxel.
Pharmacists working in the intensive care unit (ICU) in Saiseikai Yokohamashi Tobu Hospital are mainly responsible for managing the stock of drugs, providing drug information to other medical staff, educating them for rational drug therapy, and providing pharmaceutical care to the patients. In order to evaluate the contribution to the rational drug therapy, we investigated the acceptance rate of the drug information that the pharmacists in the ICU provided to the physicians from February to May in 2009. The number of cases in which drug information was provided by the pharmacists to the physicians during the period was 288. It was suggested that more than half of the information could optimize the drug dosage regimens and correct the inadequate prescriptions. Furthermore, 98.9% of the information provided by pharmacists was accepted by physicians. We questioned 5 intensivists to evaluate the information with a 5 point scale (maximum score was 4, minimum score was 0) and then the average of score was 3.3. In addition, their evaluation of the information about optimizing the drug dosage regimens marked the highest point (over 3.5). Meanwhile, providing drug information which led the physicians to correct the inadequate prescriptions contributed to reduce the cost of the drug therapy by 900000 yen during the period. As a result, it was suggested that the intensivists highly appreciated the information offered by the pharmacists and the information contributed to enhance high-quality drug therapy. Additionally, the economic impact was identified through the cost reduction in drug therapy.
Chemotherapeutic drug dosages are calculated precisely based on the patient's height, body weight, and renal function, etc. To ensure safe and favorable outcomes of treatment, dosing solutions are prepared by appropriate mixing of the drug solutions based on such calculations. The package inserts for many injectable preparations include a warning for storing the product “shielded from light.” However, there are no reports of stability assessment of a mixed product against light exposure or the residual amount of active ingredient in the dosing solution during or at the end of treatment. We evaluated the stability of carboplatin from the time of mixing of the dosing solution until the end of drug infusion in a clinical-like setting. With 4-hour exposure to outdoor scattered light, the dosing solution began to show discoloration by 1 hour, becoming dark yellow by 4 hours, with reduction of the percent residual carboplatin to about 23%. To identify the optimal light-shielding shade, the dosing solution was shielded from outdoor scattered light with 1 of 3 protective covers: aluminum foil, yellow plastic shade, and brown plastic shade. The yellow plastic shade prevented any changes of the appearance of the dosing solution during the 4-hour exposure period. The percent residual carboplatin, determined by HPLC, in the dosing solution shielded with a yellow plastic shade was about 85.2% at 2 hours and 78.6% at 4 hours. Thus carboplatin dosing solution should be completely shielded from light until infusion is completed.
We have employed a combination of cysteine mutagenesis and chemical crosslinking using a photoactivatable sulfhydryl reagent, benzophenone-4-maleimide, to obtain a covalent complex between human galectin-1 and a model glycoprotein ligand, asialofetuin. We previously obtained a crosslinked product when Lys28 of the cysteine-less form of human galectin-1 was mutated to cysteine. To investigate whether substituting either of the two flanking amino acid residues in the same β-strand, Ala27 and Ser29, to cysteine could result in crosslinking to the bound asialofetuin, two cysteine-containing mutants were generated. Although both the mutants adsorbed to asialofetuin-agarose and were eluted with 0.1 M lactose, confirming their ability to interact with asialofetuin, these mutants did not crosslink to the bound glycoprotein ligand following treatment with benzophenone-4-maleimide. Therefore the orientation of the side chain of the introduced cysteine residue apparently plays an important role in the crosslinking reaction.
The purposes of this survey were to determine the attitudes and the extent of anxiety of pregnant and lactating women about drug use, and to research priority issues for pharmacists' intervention. Postpartum lactating women and mothers with children in a Growing Care Unit (GCU) in hospitals certified as Baby Friendly Hospital (BFH) were surveyed. The questions included the images the respondents had of drugs before pregnancy, the extent of anxiety about drug use, and ways to relieve it. The highest number of respondents (49.1%) did not want to use drugs often before pregnancy, but said “physician-prescribed drugs are fine”. 24.5% had no negative images, and they “take drugs when necessary without worrying”. An additional 14.2% did not like drugs, and “avoid them whenever possible”, followed by 9.4% who did not want to use drugs, but were willing to take health food and other over-the-counter items. The respondents reported that the extent of anxiety about drug use was 79.3% during pregnancy, which was higher than 71.7% during lactation. It was not influenced by birth experience and age. “The images of drugs before pregnancy” and “the extent to which the anxiety was relieved during pregnancy” were extracted as factors related to the extent of anxiety, verifying that negative images of drugs and low degrees of relief from anxiety raise the anxiety of pregnant women. The above shows that pharmacists need to understand the anxiety of pregnant and lactating women about drug use, and the images they had of drugs before pregnancy, thereby they are expected to work actively to determine and relieve the anxiety.
We examined the effects of serum-free medium on the gene expression changes in human mesenchymal stem cells (hMSCs) during the in vitro culture using a DNA microarray analysis. In this study, we cultured hMSCs with two kinds of medium; 1) MSCGM (contain 10% fetal bovine serum) or 2) STK2 (serum-free medium developed for mesechymal stem cells multiplication), and compared hMSCs proliferation, cell morphology, and gene expression changes until 50 days culture. Expression analysis was performed with Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. hMSC proliferation was significantly higher in STK2 medium than in MSCGM medium. The cell morphology of hMSC cultured with STK2 was not significantly changed in 50 days culture. The gene expression changes in hMSCs during the in vitro culture were significantly higher in STK2 than in MSCGM. After 50 days culture, 1991 genes were significantly changed the expression levels compared with 3 days in STK2 but not MSCGM. The expressions of genes related to cell cycle, cancer, proliferation, and cell growth were significantly changed by STK2 for 50 days culture. It was also changed by STK2 that the expressions of genes related to the signaling pathways contain various growth factors, such as IGF-1, FGF, TGF-β, EGF, proliferation, and cell cycle. These results suggest that STK2 may be useful to obtain an enough number of hMSC cells for tissue engineered medical devices in short-term, however, it should be recognized that STK2 would alter the expressions of genes related to a variety of signaling pathways in hMSC if the culture period would be extended to obtain a large number of cells.