I have pursued research on lysozymes for 42 years. During that time, I made Several new findings, some of them by chance. My enjoyment of the following areas is reviewed: the story of tryptophan; protease digestion mechanisms; peptide mapping with RP-HPLC; gene engineering; renaturation of protein; catalytic residues; fluctuation and function; stabilization; folding; antigenecity; tolerance; and various lysozymes.
Crambescidins and batzelladines, novel marine guanidine alkaloids, have unique pentacyclic and tricyclic guanidine core structures, respectively. They display a considerable array of biological activity and not surprisingly have attracted considerable synthetic interest. The first total synthesis of crambescidin 359 (7) and stereoselective total synthesis of batzelladine D (11) were accomplished based on a successive 1,3-dipolar cycloaddition reaction strategy. During synthetic studies of 7, the absolute stereochemistry was revealed. Based on the structure of 7, the novel C2-symmetric pentacyclic guanidine compounds 69a—d were designed and synthesized as guanidine organocatalysts. The catalyst 69b works efficiently as an asymmetric catalyst of the alkylation reaction of the glycynate-benzophenone Schiff base 73, which gives 74 with 80—90% ee.
The antioxidant 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S) was found to be present in the placenta and to prevent the onset of preeclampsia. From experiments using rats, 2-OH-E2-17-S was confirmed to be a highly functional compound with stronger antioxidant activity than α-tocopherol and to sustain its antioxidant activity. 2-OH-E2-17-S was confirmed to be produced in the placenta from its precursor, estradiol 17-sulfate (E2-17-S), which is derived from fetal testosterone sulfate (TS). Since the fetal adrenal gland has been shown to convert testosterone (T) into TS, the following metabolic pathway may exist during pregnancy: T→TS→E2-17-S→2-OH-E2-17-S. This fetoplacental pathway may contribute to the maintenance of healthy pregnancy. Details and the experimental outline of these discoveries are reported in this review.
The oligopeptide transporter PEPT1 is predominantly expressed in the brush-border membranes of small intestinal epithelial cells, where it plays pivotal roles in the efficient absorption of di-/tripeptides. PEPT1 has enormous potential as an oral drug delivery target, because it also mediates the intestinal absorption of peptide-mimetic and nonpeptide substrates. We demonstrated that the peptide derivation of amino acid-related drugs is applicable to improve their intestinal absorption. We have found that oligopeptide transport activity is also expressed in cancer cell lines. The tissue distribution of bestatin was significantly increased in solid tumors that overexpress PEPT1 in nude mice. Orally administered bestatin strongly suppressed tumor growth. These results provided the first demonstration of the tumor-selective delivery of a drug by specific transport activity. The absolute degree of PEPT1 mRNA expression in the small intestine was determined using real-time PCR in rats. Starvation of the animals increased the mRNA expression level profoundly in the upper small intestine. The longitudinal expression pattern was well correlated with the intestinal transport of cefadroxil in rats. We constructed a recombinant adenovirus vector encoding PEPT1 cDNA. Heterologous expression of PEPT1 in the liver was successfully achieved by simple intvavenous. administration of the vector, resulting in increased liver distribution of [3H] carnosine. In situ perfusion of the brain with the vector doubled the brain distribution of cefadroxil. Heterologous expression of the drug transporter in vivo could be a useful approach for drug delivery.
Organoselenium compounds are important tools in the field of synthetic chemistry because their specific reactivities have been used as the key reactions in the synthesis of various organic compounds. Nucleophilic organoselenium reagents are frequently utilized to introduce a selenium atom into organic molecules. However, there is no method available for the direct transformation of a hydroxy group into a selenanyl group, except for the Grieco-Nishizawa reaction. In this review, novel methods for the conversion of alcohols into selenides in one step are described: both selenolate-aluminum chloride and TMSSePh-aluminum bromide are effective reagent systems for this transformation. Although benzylic alcohols could be efficiently converted to the corresponding selenides, these reagent systems were not applicable to non-benzylic alcohols. On the other hand, the reaction of cinnamyl alcohol with TMSSePh-aluminum bromide afforded 4-phenylselenochroman as a main product. The scope and limitations of this selenochroman formation were investigated using several types of allyl alcohols. To clarify the reaction path, the reaction of cinnamyl phenyl selenide with aluminum bromide was carried out. As expected, 4-phenylselenochroman was obtained in high yield. The transformation of various cinnamyl selenides was also successful in generating the corresponding selenochroman derivatives. Consequently, we developed a novel method for selenochroman synthesis.
Platycodon root, one of the most important Chinese herbal medicines, has been used as an antiphlogistic, antitussivie, and expectorant agent since ancient times. In the Japanese Pharmacopoeia XIV this is listed as the root of Platycodon grandiflorum A. De Candolle (Campanulaceae) and called KIKYOU (Platycodi Radix) in Japanese. HPLC analysis showed that commercial samples of P. Radix all contained platycodins, and a total of 12 peaks were identified by co-HPLC analysis with authentic samples isolated earlier from this laboratory. The peak purity and identity were checked with a photodiode array detector. The contents of the major saponins, platycodins A, C, and D, were determined and the peak-area ratios of platycodins A, C, and D, were shown to be correlated with their sources of origin. Fourteen commercial samples of Platycodon root, the origin of which was Platycodon grandiflorum, were collected from China (5 samples), Korea (5 samples), and Japan (4 samples). The commercial samples from China, Korea, and Japan each gave a distinct HPLC pattern with peak-area ratio of platycodins A, C, and D, of 1:2:3 and 2:8:1, respectively. HPLC analysis showed that those on the Japanese market were either imported from China or Korea based upon their HPLC patterns.
Estrogenic activities of more than 90 chemicals including food additives, foodstuffs of plant origin, and some chemicals, which could be orally ingested, were examined by assaying estrogen receptor (ER)-dependent proliferation of MCF-7 cells. Among 66 food additives, 17 compounds stimulated the proliferation, but their concentrations giving maximal cell yield were higher than that of 17β-estradiol and their estrogenic activities were weak. Flavonoids had relatively strong estrogenic activities. In the assay of ER competitive binding to human ERα and ERβ in vitro, the antioxidant t-butylhydroxyanisole (BHA) had the capacity to compete with 17β-estradiol, while the capacity of o-phenyl phenol (OPP) was too small to calculate. Both BHA and OPP induced a decrease in gene expression of ERα and an increase in that of progesterone receptor in a time-dependent manner. These effects were similar to that of 17β-estradiol, a though much higher concentrations were required for these compounds than 17β-estradiol. These results may suggest that we should be careful not to ingest excessive food additives.
We have investigated a method to analyze the marker substance of natural medicines by employing capillary electrophoretic techniques such as micellar electrokinetic chromatography (MEKC). We previously reported a simple MEKC assay method for calycosin in Astragali Radix and its fluid extract. In this investigation, we analyzed the marker substance atractylenolide III in Atractylodis Rhizoma. Atractylenolide III was clearly separated from the other components in A. Rhizoma and its dried extract by simple solvent extraction with hexane and subsequent MEKC analysis with sodium dodecyl sulfate (SDS) within 10min. Validation of the analytical procedures of the assay method was performed according to the Japanese Pharmacopoeia and International Conference on Harmonization guidelines. As a result, we confirmed that capillary electrophoresis, including MEKC, is a powerful method for estimating the quality of natural medicines and traditional Chinese medicines consisting of various components.
We have held practical classes called chozaigijutsu-konwakai(Konwakai) for pharmacists working in drugstores eve-ry month since December 1994. Konwakai is composed of lectures and practice. The practice is carried out for pharmacists to obtain a better understanding of prescriptions and compounding them in exercises. In this study, we analyzed the answers to questionnaires distributed at the end of each konwakai to all participants. It was found that the age of participants as well as the length of compounding experience varied markedly, indicating that pharmacists of various generations are interested in konwakai. Regarding the contents of lectures and practice, the answers indicated that the practice was particularly useful, suggesting the importance of exercise for learning techniques in compounding. Since there were many requests for consultations on routine work, frequent communications between pharmacists in the hospital and drugstores appears necessary. In conclusion, regular meetings including practical classes such as konwakai are very important for pharmacists in drugstores that receive prescriptions from hospitals. Further efforts to improve the konwakai should be maid in the future.
The area distribution of out-issued prescriptions for outpatients in Saiseikai Kurihashi Hospital was surveyed over 6 months from the first day of out-issue. Behavior patterns of outpatients in the selection of a pharmacy were also investigated. Our survey included the out-issued prescriptions in eight surrounding newly established pharmacies together with the first out-issue. Computer simulation indicated that 70% of the outpatients selected the surrounding pharmacies when they received their out-issued prescriptions the first time. However, the percentage of surrounding pharmacies selected by the patients decreased to 60% the second time, and then again increased to 80% the third time. The results suggest that most outpatients use surrounding pharmacies, although they try to use the pharmacies near their homes for a time.