Introduction of DPR, an enterostatin fragment peptide, into soybean beta-conglycinin alpha' subunit by site-directed mutagenesis

DPR, a fragment peptide of enterostatin (VPDPR) having hypocholesterolemic activity, was introduced into the three homologous sites, EPR, DYR, and DPI, in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified beta-conglycinin was expressed in Escherichia coli and recovered in the soluble fraction. After purification on ion-exchange HPLC, the modified beta-conglycinin was digested by trypsin to release integrated DPR. The yield of DPR from 1 mole of the modified beta-conglycinin was 1.2 mole.     

Suppressive effect of a hot water extract of adzuki beans (Vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats

A hot water extract obtained by boiling adzuki beans (Vigna angularis) to produce bean paste for Japanese cake showed inhibitory activity against alpha-glucosidase, alpha-amylase, maltase, sucrase, and isomaltase after HP-20 column chromatography. The IC(50) values for each hydrolylase were 0.78 mg/ml (alpha-amylase), 2.45 mg/ml (maltase), 5.37 mg/ml (sucrase), and 1.75 mg/ml (isomaltase). The active fraction showed potential hypoglycemic activity in both normal mice and streptozotocin (STZ)-induced diabetic rats after an oral administration of sucrose, but did not show any effect on the blood glucose concentration after glucose administration, suggesting that the active fraction suppressed the postprandial blood glucose level by inhibiting alpha-glucosidase and alpha-amylase, irrespective of the endogenous blood insulin level.     

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

Serum concentrations of androstenediol and androstenediol sulfate, and their relation to cytokine production during and after normal pregnancy

Since it is known that androstenediol (ADIOL) has potent immunoregulatory effects, changes in ADIOL levels during and after pregnancy might affect the maternal immune system. We examined serum concentrations of ADIOL and androstenediol 3-sulfate (ADIOLS) together with IFN-gamma and IL-4 production levels during pregnancy and after delivery up to 10-11 months postpartum. The subjects were 73 normal pregnant, 76 normal postpartum, and 28 normal non-pregnant women. ADIOL and ADIOLS were measured using EIA and GC/MS, respectively. The cytokine levels in the supernatant of whole-blood cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin were measured using ELISA. ADIOL levels significantly decreased compared to non-pregnant levels in the first trimester (P < 0.05) and were reversed in the third trimester (P < 0.05). After pregnancy, ADIOL levels gradually declined, and a significant decrease was observed at 10-11 months postpartum (P < 0.05). ADIOLS levels were significantly lower in the third trimester (P < 0.05) and significantly higher at the first month postpartum (P < 0.001) compared to non-pregnant women. IFN-gamma and IL-4 levels decreased during pregnancy and subsequently increased postpartum. On the other hand, we found significant negative correlations between ADIOL concentrations and production levels of IFN-gamma (P < 0.05) or IL-4 (P < 0.05). These findings suggest that ADIOL may be involved in modifying the maternal immune response during and after pregnancy.     

Rabies and other lyssaviruses

Since rabies has not been reported in Japan for nearly the past 50 years, it has been relegated to the status of a forgotten infectious disease in this country. However,in the neighboring Asian countries, Africa, America, the number fo rabies cases had not decrease but on the contrary, seen an increasing trend. In Russia and the former Soviet Union countries (CIS countries), the number of information. Between 30,000 approximately 20,000 fatal cases of rabies in both humans and animals had been reported yearly butit was thought that the number might run up to hundred of thousands.japan, Taiwan, UK, Australia and New Zealand are rabies-free countries and should be considered the exception rather than the norm. Due to the long Lull in which rabies has not occurred in Japan,people tend to forget that the disease can infect all mammals including humans, with a mortality rate of 100% after manifestation of debilitating nervous symptoms and that is one of the most dangerous zoonotic viral diseases on earth.     

Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide

Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.