Suppressive effect of polysaccharides from the edible and medicinal mushrooms,Lentinus edodes and Agaricus blazei,on the expression of cytochrome P450s in mice

To investigate the effects of lentinan from Lentinas edodes and polysaccharides from Agaricus blazei (ABPS) on the expression of cytochrome P450s (CYPs), lentinan (10 mg/kg/day) or ABPS (200 mg/kg/day) was administered to female BALB/c mice four times every other day by intraperitoneal injection. Lentinan and ABPS suppressed both the constitutive and 3-methylcholanthrene-induced CYP1A expression and ethoxyresorufin-O-deethylation activity in the liver.     

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety,and oral analgesic efficacy in rat pain models

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.     

Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Trehalose augments osteoprotegerin production in the FHs74Int human intestinal epithelial cell line

The disaccharide trehalose has been shown to inhibit both bone loss in ovariectomized mice and excessive osteoclastogenesis in lipopolysaccharide-injected mice. However, the mechanism of osteoclastogenesis inhibition by oral administration of trehalose is still unclear. We report here for the first time that a human intestinal epithelial cell line, FHs74Int, also produces osteoprotegerin (OPG) and that trehalose augments OPG production by this cell line. Thus, these results suggest that trehalose promotes the production of OPG by intestinal epithelial cells, which then acts on bone marrow cells, resulting in the suppression of osteoclastogenesis.     

Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change

The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh 29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2-39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21-25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh 29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.     

Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes

Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.     

BACE1 interacts with nicastrin

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.