Masato Umeda

Nadrin is a GTPase‐activating protein (GAP) for the rho family of GTPases that controls Ca²⁺‐dependent exocytosis in nerve endings. In this study, three novel splice variants of nadrin were identified and the variants were designated as nadrin‐102, ‐104, ‐116 and ‐126 according to their relative molecular masses. All nadrin variants share the GAP domain, coiled‐coil domain, serine/threonine/proline‐rich domain, SH3‐binding motif, and a successive repeat of 29 glutamines. Tissue distribution analyses using polyclonal antibodies that can discriminate each variant showed that the expression of nadrin‐102, ‐104 and ‐116 was dominant in neuronal tissues and correlates well with the differentiation of neurons while nadrin‐126 was strongly expressed in embryonic brain. Expression of nadrin‐116 in PC12 cells strongly inhibited NGF‐dependent neurite outgrowth and this effect was dependent on its GAP activity. In contrast, no significant effect on either cell morphology or neurite outgrowth was observed with other variants. All variants showed punctate appearance throughout the cytoplasm, while the 66‐kDa carboxyl‐terminal fragment of nadrin‐102 and/or nadrin‐116 was localized to the nucleus and its nuclear translocation was accelerated by NGF‐induced differentiation of the cells. These results suggested that nadrin variants are different in their ability to regulate rho‐mediated signaling and that, in addition to being aGTPase‐activating protein, nadrin‐102 and ‐116 have otherdistinct functions in the nucleus of the cell, implying apossible role in the cross‐talk between the cytoskeleton andthe nucleus.

Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A₂ from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09–0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-{ N -[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to ≈40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.

Effects of a novel zinc compound (polaprezinc), N-(3-aminopropionyl)-L-histidinato zinc, on the mucosal ulcerogenic and healing impairing responses induced by monochloramine (NH2Cl) were examined in rat stomach. Oral administration of NH2Cl (> 60 mM) produced severe hemorrhagic lesions in unanesthetized rat stomachs with a marked increase of thiobarbituric acid reactants (TBAR). Pretreatment of the animals with polaprezinc (3 approximately 30 mg/kg, p.o.) showed a dose-dependent inhibition against gastric ulcerogenic and TBAR responses induced by NH2Cl (120 mM). Likewise, mucosal exposure to NH4OH (60 mM) in urethane anesthetized stomachs made ischemic by bleeding from the carotid artery (1 ml per 100 g body w.t.) resulted in severe gastric lesions. This ulcerogenic response caused NH4OH plus ischemia was also attenuated by prior application of polaprezinc as well as taurine (25 mg/ml, 1 ml). On the other hand, the healing of gastric mucosal lesions induced by NH2Cl occurred more slowly than of ethanol-induced lesions, and the latter was significantly delayed by the repeated administration of NH2Cl. Polaprezinc (> 10 mg/kg, p.o.) given twice daily for 7 days not only accelerated the healing of NH2Cl-induced gastric lesions but also antagonized the delayed healing of ethanol-induced lesions in the presence of NH2Cl as well. Polaprezinc showed a scavenging action against NH2Cl in vitro. These results suggest that NH2Cl caused deleterious action on the healing of pre-existing acute lesions as well as irritating action to the mucosa in the rat stomach. Polaprezinc not only protects the stomach against injury caused by NH2Cl but also promotes healing of NH2Cl-induced gastric lesions as well as the delayed healing of ethanol-induced lesions caused by NH2Cl. Although the detailed mechanisms underlying these actions of polaprezinc remain unknown, they may be partly attributable to a scavenging action of this agent against NH2Cl.