This review documents my research for the past 45 years in peptide chemistry. Initially, in order to study the structure-activity relationships of active center of α- and β-melanocyte stimulating hormones (H-His-Phe-Arg-Trp-Gly-OH), we employed D-amino acids. That approach yielded first published report in 1965 of antagonists containing D-amino acids. Monkey β-melanocyte stimulating hormone (β-MSH), an 18 amino acid peptide stimulated pigment cells. We synthesized β-MSH and fragments thereof, and studied in detail structure-activity relationships. A major and valuable result revealed that the C-terminal pentadecapeptide of β-MSH exhibited higher MSH activity than the parent hormone providing a new question; namely, what was the role of the N-terminal tripeptide? In order to identify the novel enzyme, spleen fibrinolytic proteinase (SFP), I developed a specific chromogenic substrate, Suc-Ala-Tyr-Leu-Val-pNA, and a specific inhibitor, Suc-Tyr-D-Leu-D-Val-pNA, once again employing my D-amino acid strategy. SFP was purified by affinity chromatography using Suc-Tyr-D-Leu-D-Val-pNA as the bound ligand. The success of this approach provided me the incentive to develop a variety of potential drugs. Thus, I prepared a specific plasmin inhibitor (YO-2) and a plasma kallikrein inhibitor (PKSI-527). Next, my research developed novel opioid receptor specific opioid agonists and antagonists based on 2′,6′-dimethyl-L-tyrosine (Dmt) dimers coupled with unique pyrazinone ring as a spacer. They exhibited potent oral antinociceptive activity acting through the μ-opioid receptor. Potent μ-receptor agonists (H-Dmt-Pro-Phe/Trp- Phe-NH2) were transformed into highly selective μ-receptor antagonists (N-allyl-Dmt-Pro-Phe/Trp-Phe-NH2), which reversed ethanol-induced increases in GABAergic neurotransmission, suggesting the possibility that they may emerge as candidates for the treatment of ethanol addiction.
Over 30 years, our laboratory has been involved in the search of bioactive natural products from plant sources of several plant families, Rutaceae, Guttiferae, Avicenniaceae, and so on. In this review, novel structures of acridone alkaloids, carbazole alkaloids, coumarins, depsidones, and so on isolated in our laboratory will be showed. In addition, some results of assay of biological activities of the isolated compounds also will be described.
Arsenic is ubiquitously distributed in nature throughout Earth's crust and thus the major source of exposure to this metalloid for the general population is naturally polluted drinking water from wells. In East Asia, more than 30 million people are chronically exposed to arsenic. Interestingly, the manifestations of vascular diseases caused by prolonged exposure to arsenic are consistent with those induced by impaired production of endothelium-derived nitric oxide (NO). However, no information has been available on the relation between NO synthesis and chronic arsenic poisoning in humans. A cross-sectional study in an endemic area of chronic arsenic poisoning in Inner Mongolia and experimental animal studies indicated that long-term exposure to arsenic by drinking water causes reduction of NO production in endothelial cells. Subsequent examinations with rabbits showed that decreased NO production during arsenic exposure is, at least in part, due to an “uncoupling” of endothelial NO synthase evoked by decreased levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), a cofactor of the enzyme, leading to endothelial dysfunction. Furthermore, an intervention study in the area of chronic arsenic poisoning in Inner Mongolia suggested that decreased NO levels and peripheral vascular disease in arsenosis patients can be reversed by exposure cessation. In our cellular experiments, we found that arsenic exposure causes adaptive responses against oxidative stress and arsenic cytotoxicity through Nrf2 activation. This review summarizes the results of our recent studies on a fusion of field and laboratory studies on the chronic arsenic poisoning and cellular protection against the metalloid.
Oxidation is synthetically important as functional group transformation as well as bond-forming method. Hypervalent iodine reagents such as phenyliodine (III) diacetate (PIDA) and phenyliodine (III) bis (trifluoroacetate) (PIFA) are one of the promising reagents for development of environmentally benign oxidations, due to their mild reactivity, low toxicity, safety, ready availability, and easy handling. Our recent challenge to this chemistry is to enhance the synthetic value of the reagents as useful alternative to highly toxic heavy metal oxidants and even rare transition metals, by pioneering a new method for enabling their efficient utilization and development of unique and new reactivities. During this course, we have succeeded in the development of new recyclable reagent 1 and its catalytic utilization, and design of a new chiral reagent 2 and its application to asymmetric oxidations. The summary of these important achievements in hypervalent iodine (III) chemistry is described herein.
This review describes our recent efforts in the development of acid-base organocatalysts, (S)-3-(N-isopropyl-N-3-pyridinylaminomethyl) BINOL (6a) and (S)-3-[2-(diphenylphosphino)phenyl] BINOL (13a), with dual activation mechanism for the aza-Morita-Baylis-Hillman (aza-MBH) reaction. In these catalysts, chiral Brønsted acid units are connected with a Lewis base unit via a spacer. The acid-base moieties act cooperatively as an enzyme-mimetic catalyst to activate substrates in the carbon-carbon bond forming reaction between enones and N-tosylimines with high enantioselectivity.
Circulatory failure in septic shock is due to vascular hyporesponsiveness, in which a massive amounts of nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. In response to various inflammatory stimuli, prostanoids are also derived from inducible isoform of cyclooxygenase-2 (COX-2). Several reports on the cross talk between NO and prostanoids have been published; vasodilator prostanoids such as prostacyclin (PGI2) and prostaglandin E2 enhance iNOS expression in cultured cells. However, the details of the cross talk between prostanoids and the iNOS-NO system remains unknown. We examined inflammatory cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the thromboxane A2 (TXA2) receptor (TP-/- mice). The cytokine-induced iNOS expression and NO production were significantly augmented in TP-/- VSMCs. Furthermore, U-46619, a TP agonist, inhibited the cytokine-induced iNOS expression and NO production. The cytokine-induced hyporesponsiveness of aortas to vasoconstrictor was significantly augmented in TP-/- aorta. Finally, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo in wild-type mice, however, this effect was not observed in TP-/- mice. These results suggest that TXA2 has a protective role against the development of the vascular hyporesponsiveness via its inhibitory action on iNOS-NO system under pathological conditions such as sepsis. Thus, it seems that the cross-talk between PG and NO works to maintain the vascular homeostasis in the systemic inflammatory reactions such as sepsis.
Cerebral ischemia, a pathological condition in which brain tissue experiences a shortage or lack of glucose and oxygen, provokes an irreversible neurodegenerative disorder that may lead clinically to a progressive dementia and global cognitive deterioration. Accumulating evidence indicates many biochemical cascades that lead ultimately to ischemia-induced cell death. However, the cellular and molecular aspects of cerebral ischemia are not yet fully understood. Since the pattern of pathophysiological alterations is not the same for all cells in the ischemic brain, a good understanding of the cellular and molecular alterations induced by cerebral ischemia is needed to develop strategies for the treatment of stroke. This review summarizes recent advances concerning the pathophysiological alterations caused by cerebral ischemia, focusing on the modification of properties of glutamate receptors, which modification may be linked to the development of cerebral infarction. Furthermore, the effects of hepatocyte growth factor on learning dysfunction and cerebral vessel injury after cerebral ischemia are also summarized. Finally, this review describes a possible ameliorative effect of the injection of exogenous neural progenitor cells on cerebral ischemia-induced learning and memory dysfunction.
Dormancy in an organism is an adaptive response to environmental stress. The initiation, maintenance, and breaking of dormancy are adaptations to environmental signals. In active cells, an environmental response is genetically controlled general phenomenon. However, no such system is available in dormant cells, in which almost all gene expression is repressed. Bacterial spores are dormant and highly resistant to many environmental stresses. I analyzed the protein profile of Bacillus subtilis spores by a combination of SDS-PAGE and LC-MS/MS to reveal protein modification in dormant cells. I found that protein modification was mediated by spore built-in enzymes YabG (a protease) and Tgl (a transglutaminase) in the spores of B. subtilis. The rearrangement of spore coat proteins caused by the activities of these built-in enzymes proceeded independently of gene expression or de novo protein synthesis in dormant cells. The results suggest that some built-in enzymes are activated under certain conditions and thereafter become involved in the modification of proteins and other cellular materials in dormant cells. I propose the idea that “Active” adaptation in active cells is dependent on gene expression, and that “Quiet” adaptation in dormant cells is dependent on the activity of some built-in enzymes independently of gene expression or de novo protein synthesis. Other enzymes are involved in restoration of dormancy in response to signals such as the nutrition.
In a pathological mechanism of Alzheimer's disease (AD), amyloid β peptide (Aβ) 1-42 plays a crucial role. However, the detailed pathological mechanism remains unclear. This elucidation is hampered by handling difficulties of Aβ1-42 due to its poor water-solubility and uncontrollable aggregation. These properties prevent reproducing neurotoxicity-related assembly events of Aβ1-42 in the experiments, leading to discrepant study outcomes. Namely, such properties of Aβ1-42 are serious obstacles to establish an experiment system that clarifies the pathological mechanism of Aβ1-42 in AD. To solve these problems, we developed “click peptide” of Aβ1-42 based on the “Ο-acyl isopeptide method”. The click peptide, which contains an Ο-acyl instead of N-acyl residue at Gly25-Ser26 of Aβ1-42, is converted to Aβ1-42 via an Ο-to-N intramolecular acyl migration upon being triggered by pH-change (pH-click) or photo-irradiation (photo-click). The click peptide was 100-fold more water-soluble than Aβ1-42 and clearly adopted a monomeric random coil structure due to the Ο-acyl moiety in the peptide backbone. The click peptide was quickly converted to monomer Aβ1-42 with a random coil structure under physiological conditions upon an action (click). The obtained Aβ1-42 underwent both self-assembly and conformational changes with time. Because the in situ production of intact Aβ1-42 from the water-soluble and non-aggregative precursor could overcome the handling problems of Aβ1-42, this click peptide strategy would provide a reliable experiment system to investigate the pathological functions of Aβ1-42 in AD.
Bacillus cereus is one that causes of opportunistic human infections. Sphingomyelinase produced by B. cereus is assumed a virulence factor for the infection. Sphingomyelinase from Bacillus cereus (Bc-SMase) is Mg2+-containing metalloenzyme. Bc-SMase is a family of neutral SMase (nSMase) and mimics the actions of the endogenous mammalian nSMase in causing differentiation, development, and apoptosis. Bc-SMase may be a good model for the poorly characterized mammalian nSMase. Activation of Bc-SMase by divalent metal ions was in the order Co2+>Mn2+>Mg2+≫Ca2+>Sr2+. Crystal structure analysis of Bc-SMase bound to Co2+, Mg2+, or Ca2+ revealed that the water-bridged double divalent metal ions at the center of the cleft in both the Co2+- and Mg2+-bound forms is the catalytic architecture required for sphingomyelinase activity. In contrast, the architecture of Ca2+ binding at the site showed only one binding site. A further single metal-binding site existed at one side edge of the cleft. Based on the highly conserved nature of amino acid residues of the binding sites, the crystal structure of Bc-SMase with Mg2+ or Co2+ provided a common structural framework applicable to phosphohydrolases belonging to the DNase I-like folding superfamily. In addition, our analysis provided evidence that β-hairpin containing the aromatic amino acid residues and the metal ion of the side-edge participate in binding to sphinogmyelin and membranes containing sphingomyelin. This article summarized current knowledge of characteristics and mode of action of Bc-SMase.
Epalrestat (Kinedak®) is an aldose reductase inhibitor (ARI) for diabetic peripheral neuropathy. In 41 diabetics, we conducted a questionnaire survey to evaluate symptoms of peripheral neuropathy and select appropriate drug therapy. We investigated 27 patients who participated in the first and second questionnaire surveys. We reviewed questionnaire items, and examined the correlation between the therapeutic effects and responses to the questionnaire. Concerning the usefulness of the questionnaire items, some questions were correlated with the effects. Treatment was effective for somatic neuropathy, but not for autonomic neuropathy. The questionnaire regarding diabetic peripheral neuropathy was useful for somatic neuropathy screening, but it was difficult to detect autonomic neuropathy.
The acid property of alkali and alkali earth cation exchanged clinoptiolites were observed by micro-calorimetry of NH3 adsorption at 200°C. The reaction rates on decomposition of tert-butyl acetate (TBA) over clinoptilolites was proportional to the acid strength. 1,4-Dihydropyridines were oxidized to corresponding Pyridines in high yields at room temperature by H2O2 aqueous solution over Mg2+ ion exchanged clinoptilolte (CZ-Mg) in acetone. Solventless acid ester decomposition of Di-tert-butyl 3,5-pyridinedicarboxylate to 3,5-Pyridinedicarboxylic acid was effected using CZ-Mg at 170°C.
Heparin is widely used as an anticoagulant for the treatment and prevention of thrombotic disorders. Recently, hundreds of cases of anaphylactic reaction as adverse effects were reported by the presence of contaminating oversulfated chondroitin sulfate (OSCS) in some heparin preparations. In addition, these heparin preparations often contaminated dermatan sulfate (DS). Unfortunately, the Japanese Pharmacopoeia (JP) does not include appropriate purity tests. In the present paper, we show that capillary electrophoresis (CE) is a powerful tool for the analysis of OSCS and DS in heparin preparations. CE method shows high resolution and good quantification of OSCS in heparin preparations. This method (OSCS method) was evaluated for accuracy (93.7 %), repeatability (R.S.D.=2.11), linearity (R2=0.9996), detection limit (0.1% OSCS) and specificity. In contrast, DS was not able to be detected in high sensitivity by OSCS method. However, a modified CE method (DS method) using the buffer at lower pHs showed good parameters for accuracy (88.1%), repeatability (R.S.D.=1.99), linearity (R2=0.9998), detection limit (0.25% DS) and specificity. In conclusion, CE will be an alternative to the NMR method which is being adopted for purification test of heparin sodium in the present version of JP.
In order to estimate the 4th grade-students' knowledge regarding clinical trials, we carried out the questionnaire survey to the students being about to take practice in hospitals, and could collect answers from 163 students. This survey revealed that about 25% of the students could not draw the difference between “clinical trial” and “clinical research”. As for the question about the technical terms regarding the clinical trial, clear correlation between students' learning experience and their knowledge was suggested. Furthermore, over 46% of the students answered that the pharmacists acting as clinical research coordinator (CRC) could not recruit new subjects, and ca. 40% of the students answered that the pharmacists acting as CRC could prescribe new drug. These misunderstandings seemed to result in the fact that most of the students could not have any chance to actually watch CRC's work related to the university curriculum. Based on the findings described above, sharing the information on the clinical trial between hospitals and universities seemed to be required to deepen students' understanding.