YAKUGAKU ZASSHI, 130(5),629-630, 2010
YAKUGAKU ZASSHI, 130(5),631-640, 2010
The Targeted Protein Research Program (TPRP) started in 2007 as a sequel of the Protein 3000 Project which lasted from 2002 to 2007. In the new project, four cores, Protein Production, Structure Analysis, Control of Protein Functions with Compounds, and Informatics, have been established as focus of methodology developments critical for functional and structural studies by the target protein research teams. Within the “Analysis Core” synchrotron radiation plays a pivotal role providing X-ray beams for structural analyses of the target proteins. The two large Japanese synchrotron radiation facilities, SPring-8 and Photon Factory (PF), along with three protein crystallography groups from Hokkaido, Kyoto and Osaka Universities have teamed up to develop two complementary micro-beam beamlines, one on each synchrotron site, and associated technologies for cutting edge structural biology research. At the PF, there are 5 operational beamlines which are equipped with state-of-the-art instrumentation for high-throughput protein crystallography experiments. Within the TPRP framework, the PF is developing a micro-focus beamline optimized for a lower energy single anomalous diffraction (SAD) experiment. This will be particularly useful for structure determination of difficult protein targets for which heavy atom derivatives or selenomethionine substitution does not work and other standard phasing methods fail to give structure solutions. This will augment the capabilities of the PF structural biology beamlines with similar look-and-feel experimental environments.
Key words--Targeted Protein Research Program; protein crystallography; Photon Factory
YAKUGAKU ZASSHI, 130(5),641-648, 2010
Recently, researchers' demands for high-quality data collection from protein micro-crystals, which would usually result from crystallization difficulty, have been remarkably growing. In order to collect efficient diffraction data for structure determination from micro-crystals, an expert data collection system providing high signal-to-noise ratio should be essential. A small sized and highly brilliant X-ray beam with size of a few micrometers has been proved to achieve protein micro-crystallography by both increasing reflection intensities and reducing background scattering from sample environments. Two micro-beam beamlines at SPring-8 and KEK-PF are currently under construction for Targeted Proteins Research Program by MEXT of Japan. At SPring-8, a new undulator beamline for protein micro-crystallography, named RIKEN Targeted Proteins Beamline (BL32XU), will start operation in May, 2010. The beam size at sample position of BL32XU will correspond to 1×1 μm2 with the photon flux density over 6×1010 photons/s/μm2. Furthermore, research and developments of indispensable components to achieve protein micro-crystallography with micro-beam, such as high-precision diffractometer, automated micro-crystal handling, advanced data collection system to suppress radiation damage and so on, are progressing. The present status of the research and development for protein micro-crystallography will be presented.
Key words--protein micro-crystallography; synchrotron radiation; SPring-8
YAKUGAKU ZASSHI, 130(5),649-655, 2010
Nowadays, three-dimensional structure of protein becomes important to understanding and application of molecular mechanisms in enzyme reaction, signal transduction and other various biochemical processes. The amount of the information is now growing quantitatively and qualitatively, supported in part by the technical development of macromolecular crystallography. In the development, brilliant synchrotron radiation greatly contributes to enhance the accuracy and precision of diffraction data and the throughput of data collection. At SPring-8, JASRI and RIKEN collaborate to utilize the macromolecular crystallography beamlines and actualize these improvements. High throughput and routine analysis of protein structures is achieved by the development of automation system composed of sample exchange robotics and control software. The remote data collection system using the automation system and internet technology enhances efficiency and convenience of the beamlines. Moreover, the development of a rapid-readout complementary metal oxide semiconductor (CMOS) detector will improve throughput in data collection. On the other hand, data collection with high accuracy and precision is achieved by the utilization of brilliant X-ray produced from the in-vacuum undulator. Its brightest and stable beam enables high resolution data collection and 10 μm microbeam for microcrystals. Although the high brilliance severely damages protein samples, the users can estimate the degree of the damage and plan best data collection strategy.
Key words--macromolecular crystallography; synchrotron radiation; microbeam; laboratory automation; complementary metal oxide semiconductor detector; radiation damage
YAKUGAKU ZASSHI, 130(5),657-664, 2010
Crystallography enables us to obtain accurate atomic positions within proteins. High resolution X-ray crystallography provides information for most of the atoms comprising a protein, with the exception of hydrogens. Neutron diffraction data can provide information of the location of hydrogen atoms, and is complementary to the structural information determined by X-ray crystallography. Here, we show the recent result of the structural determination of drug-target proteins, porcine pancreatic elastase and human immuno-deficiency virus type-1 protease by both X-ray and neutron diffraction. The structure of porcine pancreatic elastase with its potent inhibitor was determined to 0.94 Å resolution by X-ray diffraction and 1.65 Å resolution by neutron diffraction. The structure of HIV-PR with its potent inhibitor was also determined to 0.93 Å resolution by X-ray diffraction and 1.9 Å resolution by neutron diffraction. The ionization state and the location of hydrogen atoms of the catalytic residue in these enzymes were determined by neutron diffraction. Furthermore, collaborative use of both X-ray and neutron to identify the location of ambiguous hydrogen atoms will be shown.
Key words--neutron crystallography; x-ray crystallography; drug-target protein; inhibitor; drug design
YAKUGAKU ZASSHI, 130(5),665-670, 2010
Ibaraki Prefectural Government together with Ibaraki University and Japan Atomic Energy Agency (JAEA) has almost finished constructing a time-of-flight (TOF) neutron diffractometer for biological macromolecules for industrial use at J-PARC, IBARAKI Biological Crystal Diffractometer (iBIX). Since 2009, Ibaraki University has been asked to operate this machine in order for users to do experiments by Ibaraki Prefecture. The diffractometer is designed to cover sample crystals which have their cell edges up to around 150 Å. It is expected to measure more than 100 samples per year if they have 2 mm3 in crystal volume, and to measure even around 0.1 mm3 in crystal volume of biological samples. The efficiency of iBIX is also expected about 100 times larger than those of the present high performance diffractometers at JRR-3 in JAEA when 1MW power realizes in J-PARC. Since December 2008, iBIX has been open to users and several proteins and organic compounds were tested under 20 kW proton power of J-PARC. It was found that one of their proteins was diffracted up to 1.4 Å in d-spacing, which was nearly comparable resolution to that of BIX-3 in JRR-3 when used the same crystal as at iBIX for reasonable exposure time. In May 2009, 14 detector units were set up. By the end of fiscal year 2009, the basic part of data reduction software will be finished and an equipment blowing low temperature gas to the sample will be installed with the cooperation of JAEA.
Key words--neutron protein crystallography; J-PARC; industrial use; protonation; hydrogen; hydrated water
YAKUGAKU ZASSHI, 130(5),671-672, 2010
YAKUGAKU ZASSHI, 130(5),673-678, 2010
Among a variety of food materials, some are being used as resources of traditional, alternative, and/or complementary medicines all over the world. These medicinal foods are known to have not only nutritive and taste values but also medicinal effects, and they are prescribed in various traditional preparations. Regarding this point, we focused on exploring bioactive constituents in these medicinal foods, which would be applicable to remedy so-called metabolic syndrome. In this review, our recent studies on anti-hyperlipidemic saponin constituents from flowers of Bellis perennis are described.
Key words--pharmaceutical food science; medicinal food; Bellis perennis; anti-hyperlipidemic activity; antiobesity
YAKUGAKU ZASSHI, 130(5),679-686, 2010
Recent progress in glycobiology reveals that sugar-chains play a crucial role in cell-cell recognition among immunity, inflammation, and malignant tumor in the living body. To study the mechanism of action requires a sample of sufficient quantity. However, isolating a sugar chain and a glycoside in pure form, difficulty follows sugar chain composition again, and studies have been limited to a few sugar chains. Glycosides are a group of compounds known to be the active principle of a natural drug. We have isolated triterpene glycosides from a Leguminosae plant, which improved liver disorder, and steroid glycosides from a plant of the Solanaceous with cytotoxic activity against human cancer cell lines. A biological activity test suggested an important role of the aglycone part and the sugar chain part of those glycosides. Although many studies of sugar chains of glycoprotein and glycolipid are known, there are few examples of studies of the sugar chain function of a glycoside of a natural drug, and the role of a sugar chain of a glycoside for its pharmacologic action expression is unknown. Therefore, as a biological tool investigating the function of a sugar chain of a natural glycoside, we have begun to synthesize a useful “glycoside” which can be utilized as a lead compound having activity.
Key words--glycoside; Leguminosae; Solanaceae; triterpenoid, steroid, cytotoxic activity
YAKUGAKU ZASSHI, 130(5),687-695, 2010
The research field of pharmacognosy covers a wide variety of subjects based on a knowledge of natural medicines and extends its influence in all surrounding directions. Pharmacognosy itself is generally regarded as one of the branches of pharmaceutical science although the processes to achieve the objectives are not always analytical as are other branches in this field. The extraordinarily long history and broad view provide researchers opportunities to conduct unique projects which can enhance the perspective of the subject in the future. Fieldwork is one feature of scientific research and is viewed as more valuable to projects in pharmacognosy than to those in other fields of pharmaceutical sciences. The frame of my research has been formed around fieldwork; projects targeted are achieved based on information and samples gathered through this means, and further studies are suggested by the ideas obtained. Some of the results and experiences gathered in these projects are described in this article for discussion of the future image of the pharmacognosial field.
Key words--pharmacognosy; fieldwork; perilla; agarwood; cinnamon
YAKUGAKU ZASSHI, 130(5),697-697, 2010
YAKUGAKU ZASSHI, 130(5),699-705, 2010
We have previously found that antitussive drugs inhibit G protein-coupled inwardly rectifying potassium (GIRK) channel currents in brain neurons. Potassium efflux through GIRK channels causes membrane hyperpolarization, and thus plays an important role in the inhibitory regulation of neuronal excitability. Because GIRK channels are coupled to various G protein-coupled receptors including monoamine receptors, antitussives are possible to affect the levels of various neurotransmitters in the brain. Many currently available antidepressants have been developed based on the monoamine theory for the etiology of depression. We hypothesized that new drugs such as tipepidine may lead to changes in the balance of monoamine levels in the brain resulting in improvement in symptoms of depression. Therefore, we investigated whether or not the drugs have antidepressant activity in the animal models. Male Wistar rats (200-240 g) were used. Tipepidine, cloperastine and caramiphen significantly reduced the immobility in forced swimming test (FST) using normal rats. All drugs had little effect on loco-motor activity. The effects on the forced swimming were inhibited by treatment with AMPT, but not PCPA. Tipepidine also inhibited hyperactivity in olfactory bulbectomized rats. Interestingly, tipepidine also significantly reduced the immobility in FST using ACTH-treated rats which is a model of depression resistant to treatment with antidepressants. Given these results together with cumulated findings, it is suggested that tipepidine may have a novel antidepressant-like action, and that the effect may be caused at least partly through the action on the catecholaminergic system in the brain.
Key words--antitussives; forced swimming test; monoamine; G protein-coupled inwardly rectifying potassium channel; microdialysis method; antidepressant-like action
YAKUGAKU ZASSHI, 130(5),707-712, 2010
Fruit juice of Morinda citrifolia (Rubiaceae, Noni juice) is a well-known healthy drink and has various pharmacological properties including antioxidant and anti-inflammatory effects. We have hitherto found the protective effect of Noni juice on neuronal damage caused by ischemic stress in mice. In addition, we have also recently reported that suppression of post-ischemic glucose intolerance might be important for good prognosis. Here, we focused on the effect of Noni juice on the development of the post-ischemic glucose intolerance as a cerebral protective mechanism. Noni juice was obtained from the mature fruit grown in Okinawa (ONJ). Male ddY mice were given 10% ONJ in drinking water for 7 d. Then, mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). We found that 10% ONJ treatment suppressed the development of neuronal damage after MCAO. Interestingly, glucose intolerance observed on 1 d after MCAO completely disappeared by 10% ONJ. Furthermore, 10% ONJ treatment significantly increased serum insulin levels much further than the control group on 1 d, while serum adiponectin levels were not affected at all. These results suggest that ONJ could facilitate insulin secretion and may attenuate the development of glucose intolerance under ischemic stress. These functions may contribute to the neuronal protective effect of ONJ against ischemic stress.
Key words--cerebral ischemia; Morinda citrifolia; glucose intolerance; insulin sensitivity
YAKUGAKU ZASSHI, 130(5),713-715, 2010
We measured both serum and saliva caffeine concentration using HPLC and assessed the correlation between them in volunteers with mild obesity. Significant correlation was shown between saliva and serum caffeine concentration. It may be necessary to measure caffeine metabolite concentration because its metabolites may also have an improving effect of glucose metabolism. In summary, we found that saliva caffeine concentration measurement was useful to assess caffeine intake level. Moreover, it will be helpful to know whether caffeine has an improving effect of glucose metabolism.
Key words--caffeine; glucose metabolism; HPLC; saliva; serum
YAKUGAKU ZASSHI, 130(5),717-721, 2010
In the brain of Alzheimer's patients, the cholinergic neurons innervated the hippocampus and cerebral cortex degenerates before accumulation of beta-amyloid protein. Donepezil, a potent acetylcholinesterase (AChE) inhibitor is reported to rescue neurons from excitotoxic injury in culture. However, there is no evidence to confirm its neuroprotective effect on ACh neurons in vivo. Using olfactory bulbectomy (OBX) mice, we defined the neuroprotective mechanisms of donepezil on the medial septum cholinergic neurons with concomitant improvement of the impaired cognitive function. Bilateral olfactory bulbs of DDY mouse were removed by surgery. After olfactory bulbectomized (OBX), donepezil (1 or 3 mg/kg/day) was administered for 15 days and mouse brain was fixed with paraformaldehyde perfusion at day 18. Then, the neuroprotective effect of donepezil was evaluated by counting the number of Chdine acetyltrans-ferase (ChAT) immunoreactive neurons in the medial septum. The number of ChAT immunoreactive neurons in the medial septum reduced by 40% of that in sham-operated animals. The reduced ChAT positive neurons were restored by donepezil treatments. Consistent with these observations, the cognitive deficits observed in OBX mice were significantly improved by the donepezil treatment. Taken together, donepezil treatment rescues the cholinergic neurons in the medial septum from the neurodegeneration by OBX. We will also discuss the mechanism underlying the donepezil-induced neuroprotection in the medial septum cholinergic neurons.
Key words--neurogenesis; cognitive function; donepezil; Alzheimer's disease
YAKUGAKU ZASSHI, 130(5),723-728, 2010
It is widely accepted that vascular endothelium regulates vasoconstriction via release of endothelium-derived relaxing factors (EDRF). The mesenteric circulation, which is the largest vascular bed, influences regulation of systemic blood pressure. However, the role of EDRF in the modulation of vascular tone in peripheral mesenteric circulation has not been extensively studied. Therefore, our recent studies investigated the role of the vascular endothelium in the regulation of methoxamine (α1-adrenoceptor agonist)-induced vasoconstriction and their age-related changes in rat mesenteric vascular beds. In mesenteric vascular beds with intact endothelium isolated from 8 week-old rats, the initial maximum vasoconstriction induced by continuous perfusion of methoxamine was time-dependently decreased during 3 hour-perfusion. Neither nitric oxide synthase inhibitor nor cyclooxygenase inhibitor altered this time-dependent reduction of methoxamine-induced vasoconstriction. Endothelium removal, K+-channel inhibitors and gap junction inhibitor significantly inhibited the time-dependent reduction of methoxamine-induced vasoconstriction. In the preparations with intact endothelium from 16 week-old rats, the time-dependent reduction of methoxamine-induced vasoconstriction disappeared. Furthermore, endothelium removal and treatment with cyclooxygenase inhibitor, thromboxane A2 receptor antagonist or superoxide dismutase mimetic significantly reduced the methoxamine-induced vasoconstriction in the preparations from 16 week-old rats. These findings suggest that vascular endothelium acts to depress methoxamine-induced vasoconstriction by releasing endothelium-derived hyperpolarizing factor (EDHF), and dysfunction in this endothelial modulation develops with ageing.
Key words--agonist-induced vasoconstriction; endothelium-derived hyperpolarizing factor; ageing; mesenteric resistance artery
YAKUGAKU ZASSHI, 130(5),729-735, 2010
A well known superdisintegrant like croscarmellose sodium or crospovidone loses their quick disintegration property when compressed at more than 4 kg tablet crushing strength (TCS). Therefore, the objective of the present work was to develop a disintegrating system that could be used for preparing fast disintegrating tablets (FDTs) of highly water soluble drug, metoclopramide, without compromising on the mechanical strength, irrespective of the TCS used for compressing the granules. For this purpose disintegrating system consisting of chitosan-alginate (CTN-ALG) complex (1:1): glycine and chitin was developed. The results revealed that when CTN-ALG and glycine were mixed in the ratio of 30:70, the granules exhibited a minimum water sorption time and maximum effective pore radius (Reff.p). The addition of chitin (5-10% w/w) into this mixture further enhanced the Reff.p. Further, increase in the concentration of chitin from 10% to 20% w/w did not produce any significant effect (p>0.05) on the Reff.p. The FDTs prepared by using CTN-ALG:glycine (30:70) and chitin exhibited increased porosity and lower disintegration time (DT). Further, chitin was found to neutralize the effect of TCS on DT of FDTs. This property of chitin was also observed in FDTs prepared by using croscarmellose sodium (5% w/w) or crospovidone (5% w/w). The reduction in DT of FDTs by chitin was also observed in tablets prepared without the drug. Hence, the effect was not influenced by the solubility component present in the tablets. Overall, the results suggested incorporation of chitin (5-10% w/w) while preparing FDTs of metoclopramide to enhanced the disintegration without compromising their mechanical strength of the tablets.
Key words--fast disintegrating tablet; glycine; chitosan-alginate complex; chitin; metoclopramide HCl
YAKUGAKU ZASSHI, 130(5),737-746, 2010
Neurodegenerative disorders, such as Alzheimer's disease (AD), is associated with the loss of neuronal cells, and it has been suggested that apoptosis is a crucial pathway in neuronal loss in AD patients. Recent evidence suggests that amyloid beta peptide (Aβ) induces neuronal apoptosis in the brain and in primary neuronal cultures. In this study, we investigated the impact of β-asarone against the apoptosis induced by Aβ in rat hippocampus. The results showed that intrahippocampal injections of Aβ (1-42) caused apoptosis in rat hippocampus. Oral administration of β-asarone (12.5, 25, or 50 mg/kg) for 28 d reverse the increase in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive cells in the hippocampus tissue. Mitochondrial dysfunction is a hallmark of β-amyloid (Aβ)-induced neuronal toxicity in AD. Therefore, we investigated nuclear translocation of apoptosis induction factors. Our results showed that β-asarone afforded a beneficial inhibition on both mRNA and protein expression of Bad, Bax, and cleavage of caspases 9 in rat hippocampus following intrahippocampal injections of Aβ (1-42). Our further investigation revealed that ASK1, p-MKK7, and p-c-Jun were significantly decreased after β-asarone treatment, implicating that the modulation of ASK1/c-JNK-mediated intracellular signaling cascades might be involved in therapeutic effect of β-asarone against Aβ toxicity. Taken together, these results suggest that β-asarone may be a potential candidate for development as a therapeutic agent for AD.
Key words--Alzheimer's disease; apoptosis; β-asarone; β-amyloid; c-Jun
YAKUGAKU ZASSHI, 130(5),747-754, 2010
Alcohol based hand rubs have been used for hand hygiene in health-care settings. Compared with hand scrubbing, using suitable alcohol based hand rub provides several advantages like usability in a ward with no tap, requiring less time and mildly-irritating. Alcohol provides immediate activity, but poor virucidal activity against certain viruses including norovirus. It is important to develop further improved alcohol based hand rubs which have characteristics of sufficient effectiveness, skin-safe and extended spectrum to non-enveloped viruses for infection control. In the study, in vitro microbicidal evaluations and in vivo efficacy evaluation study were investigated to clarify the characteristics of a novel hand antiseptic MR06B7 composed of additives with synergetic activities. MR06B7 showed bactericidal activity of more than 5 Log10 reduction within 15 sec against 20 challenged strains. MR06B7 also demonstrated potent fungicidal activities at exposure time of 30 sec (more than 4 Log10 reduction). Against all test viruses including non-enveloped viruses (adenovirus, feline calicivirus, murine norovirus and poliovirus), MR06B7 had excellent virucidal activity to reduce the titer of viability to the limit of detection within 30 sec exposure (more than 4 Log10 reduction), whereas 83%(v/v) ethanol indicated the inadequate effectiveness. On the clinical study conducted in accordance with standard method for Healthcare Personnel Handwash of American Society for Testing and Materials, MR06B7 showed excellent immediate antimicrobial activity. The result surpassed the critical indices set forth in the FDA's Tentative Final Monograph. These findings suggest MR06B7 which satisfies most requirements of efficacy qualifications including potent virucidal activity against non-enveloped viruses may contribute to accomplish advanced infection control in clinical practice.
Key words--alcohol; hand hygiene; virucidal activity; feline calicivirus; murine norovirus
YAKUGAKU ZASSHI, 130(5),755-761, 2010
Monovalent bile acids, such as taurine- and glycine-conjugated bile acids, are excreted into bile by bile salt export pumps (BSEP, ABCB11). Human BSEP (hBSEP) is physiologically important because it was identified as the gene responsible for the genetic disease: progressive familial intrahepatic cholestasis type 2 (PFIC-2). The evaluation of the inhibitory effect of hBSEP transport activity provides significant information for predicting toxic potential in the early phase of drug development. The role and function of hBSEP have been investigated by the examination of the ATP-dependent transport of radioactive isotopically (RI)-labeled bile acid such as a tritium labeled taurocholic acid, in membrane vesicles obtained from hBSEP-expressing cells. The chemiluminescence detection method using 3α-hydroxysteroid dehydrogenase (3α-HSD) had been developed for a simple analysis of bile acids in human biological fluids. This method is extremely sensitive and it may be applicable for the measurements of bile acid transport activities by hBSEP vesicles without using RI-labeled bile acid. The present paper deals with an application of the chemiluminescence detection method using 3α-HSD with enzyme cycling method to the measurement of ATP-dependent transport activities of taurocholic acid (T-CA) in membrane vesicles obtained from hBSEP-expressing Sf9 cells. Calibration curves for T-CA was linear over the range from 10 to 400 pmol/ml. The values of the kinetic parameters for hBSEP vesicles obtained by the chemiluminescence detection method were comparable with the values of that obtained by liquid chromatography-mass spectrometry method. This assay method was highly useful for the measurements of bile acid transport activities.
Key words--bile acid; bile salt export pump; chemiluminescence detection
YAKUGAKU ZASSHI, 130(5),763-768, 2010
The international standard ISO 8124-3: 1997 “Safety of toys -Part 3: Migration of certain elements” and “Interim Enforcement Policy for Children's Metal Jewelry Containing Lead- 2/3/2005” by the U.S. Consumer Product Safety Commission (CPSC) to control the amount of eluted lead from metal accessories cannot be simply compared, because the acid extraction methods and the limit values are different from each other. Therefore acid extraction tests based on the ISO standard and the CPSC policy were conducted and the amounts of eluted lead from small metal products were compared between both tests. There was less eluted lead in the ISO method than in the CPSC method. Moreover, the amount of eluted lead in the ISO method did not even reach that of the first elution in the CPSC method. It became clear that the acid extraction test of the ISO standard was not as good as that of the CPSC policy, because of the difference in test conditions. In 16 small metal products, seven products were unsuitable for the ISO standard and 14 products were unsuitable for the CPSC policy; however, all these products were originally inapplicable to the ISO standard and the CPSC policy. The calculation grounds of the limit values were also different between the ISO standard and the CPSC policy. The standardization of an acid extraction test that simulates the lead elution to gastric juice is required so as to prevent adverse health effects in children due to their accidental ingestion of small metal products containing lead.
Key words--lead; acid extraction test; metal product; capillary electrophoresis