Ornithine decarboxylase antizyme is a unique inhibitory protein induced by polyamines and involved in the regulation of ornithine decarboxylase. A cDNA was isolated from a rat liver cDNA library by the screening with monoclonal antibodies to rat liver antizyme as probes. The expression products of the cDNA in bacterial systems inhibited rat ornithine decarboxylase activity in a manner characteristic of antizyme and rabbit antisera raised against its direct expression product reacted to rat liver antizyme, confirming the authenticity of the cDNA. On RNA blot analysis with the cDNA probe, an antizyme mRNA band of 1.3 kb was detected in rat tissues. Antizyme mRNA did not increase upon administration of putrescine, an inducer of antizyme, and its half-life after actinomycin D treatment was as long as 12 h in rat liver, suggesting that antizyme mRNA is constitutively expressed and antizyme synthesis is regulated at the translational level. Similar-sized mRNAs hybridizable to the cDNA were also found in various mammalian and non-mammalian vertebrate tissues under physiological conditions. In addition, chicken and frog antizymes showed immunocrossreactivity with rat antizyme. The ubiquitous presence and the evolutionally conserved structure of antizyme in vertebrate tissues suggest that it has an important function.

We found previously that dipeptide YL exhibits orally active anxiolytic activity comparable to diazepam. The YL sequence is often observed in the primary structure of natural food proteins. In the present study, we investigated whether YL and YL analogues are released from bovine αS-casein by gastrointestinal proteases. YLG, corresponding to αS1-casein (aa 91-93), was more effectively released from αS-casein than YL by pepsin-pancreatin digestion, mimicking gastrointestinal enzymatic conditions. Using the synthetic model peptide, we determined that trypsin cleaved the N terminus of YLG, and elastase and carboxypeptidase contributed to cleave the C-terminus. YLG exhibited orally active anxiolytic-like activity in the elevated plus maze and open-field tests in mice. The anxiolytic-like activity of YLG was inhibited by WAY100135, SCH23390 or bicuculline, antagonists of serotonin 5-HT1A, dopamine D1, and GABA(A) receptors, respectively; however, YLG had no affinity for these receptors. The pepsin-pancreatin digest of αS-Casein also exhibited anxiolytic-like activity. Meanwhile, anxiolytic-like activity of α-casozepine, an αS1-casein-derived decapeptide with YL sequence in the N terminus, was blocked by WAY100135, SCH23390, or bicuculline, equally to YLG and YL; however, it was not detected in the pepsin-pancreatic digest. Taken together, we found that YLG is released after pepsin-pancreatic digestion of αS-casein and exhibits potent anxiolytic-like activity via activation of serotonin, dopamine, and the GABA receptor system.

No abstract

We identify a novel, orally active antihypertensive peptide, LRA from the digest of rice bran protein.

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.

Phospholipid‐phospholipid interaction in soybean oil is described. Phosphatidylcholine was effectively removed from soybean oil by degumming (water hydration), whereas phosphatidylethanolamine and phosphatidic acid were hardly hydratable. However, the degree of their hydration increased in the presence of phosphatidylcholine. The spectrophotometric assay based on charge transfer interaction between 7,7,8,8‐tetracyanoquinodimethane and phospholipids at 480 nm was used to determine the formation of phospholipid micelles in soybean oil. The critical micelle concentrations were 0.085, 0.84 and 2.6 mM for phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, respectively. Phosphatidylcholine interacted with phosphatidylethanolamine or phosphatidic acid to form mixed micelles. The critical micelle concentrations of phosphatidylcholine‐phosphatidylethanolamine mixture and phosphatidylcholine‐phosphatidic acid mixture were 0.16 and 1.3 mM, respectively. The degree of hydration of phospholipids was related to their critical micelle concentrations. Interaction of phosphatidylcholine with phosphatidylethanolamine or phosphatidic acid was confirmed by determining the changes in the chemical shifts of 31PNMR spectra.

No abstract

Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release.