A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and pore formation in the cell membrane.

Localized spins and itinerant electrons rarely coexist in geometrically-frustrated spinel lattices. They exhibit a complex interplay between localized spins and itinerant electrons. In this paper, we study the origin of the unusual spin structure of the spinel CoV2O4, which stands at the crossover from insulating to itinerant behavior using the first principle calculation and neutron diffraction measurement. In contrast to the expected paramagnetism, localized spins supported by enhanced exchange couplings are frustrated by the effects of delocalized electrons. This frustration produces a non-collinear spin state even without orbital orderings and may be responsible for macroscopic spin-glass behavior. Competing phases can be uncovered by external perturbations such as pressure or magnetic field, which enhances the frustration.

The distribution of neurocan-like and 6B4 proteoglycan-like immunoreactivities in the rat embryo was investigated from gestational days 10.5-15.5 with monoclonal antibody 1G2 or 6B4 that immunoreacted with neurocan and 6B4 proteoglycan, respectively. In the brain region, the leptomeningeal layer in the myelencephalon, metencephalon, diencephalon or telencephalon was first stained with monoclonal antibody 1G2 at embryonic day 12.5. In the spinal cord, monoclonal antibody 1G2 stained the regions corresponding to the boundary caps (designated the boundary caps) after embryonic day 11.5 and the roof plate after embryonic day 12.5. The intensity of staining in the boundary caps reached a maximum at embryonic day 13.5, at around the time when the axons from the dorsal root ganglia reach this region. However, the points of contact of the axons with the boundary caps were hardly stained. By contrast, the roof plate was most strongly and widely stained at embryonic day 14.5, at around the time when the axons enter the spinal cord. Western blotting of preparations from the spinal cord that included the boundary caps revealed the presence of neurocan in this region. Thus, it is likely that neurocan serves as a barrier molecule to regulate the direction of axonal growth from the dorsal root ganglia. By contrast, in addition to staining of the future brain and spinal cord, monoclonal antibody 6B4 stained the trigeminal and sympathetic ganglia in the rat embryo on and after embryonic day 12.5, as well as the vestibular, facial and dorsal root ganglia after embryonic day 12.5. In studies in tissue culture, monoclonal antibody 6B4 prevented the inhibitory effects of 6B4 proteoglycan on the proliferation of PC12D cells. No immunostaining with monoclonal antibody 6B4 was observed in cells that had incorporated bromodeoxyuridine in vivo. Possible functions of 6B4 proteoglycan in the rat embryo are discussed.

Intrahippocampal injection of a subtoxic dose of kainate in mice has been shown to induce a dispersion of granule cells of the dentate gyrus, which is a characteristic morphological change often seen in human hippocampal sclerosis. In addition, it has been shown recently that such injections lead to recurrent hippocampal seizures and changes in glucose metabolism, which are reminiscent of temporal lobe epilepsy. Previous reports on human hippocampal sclerosis have shown an increase of the expression of the GluR2 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate subunits in the dispersed granule cell somata. However, no such changes have been observed so far in animal models of epilepsy with hippocampal sclerosis. In this study, the expression of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunits was examined by immunohistochemistry following intrahippocampal injection of kainate in mice and rats. In mice, such injection induced a persistent increase of GluR2 immunoreactivity in the granule cells for up to 180 days. By contrast, GluR1 immunoreactivity was transiently increased during the first four days after the injection and progressively decreased thereafter. By contrast, intrahippocampal injection of kainate in rats did not result in granule cell dispersion and no changes in GluR1 immunoreactivity or GluR2 immunoreactivity were observed. These results show that, in addition to morphological, clinical and metabolical similarities, intrahippocampal injection of kainate results in a persistent increase of GluR2 associated with granule cell dispersion, as in human hippocampal sclerosis. These data suggest the existence of common mechanisms between granule cell dispersion and regulation of GluR2 subunits associated with hippocampal sclerosis.

Fibrinogen Caracas II is an abnormal fibrinogen involving the mutation of A alpha serine 434 to N-glycosylated asparagine. Some effects of this mutation on the ultrastructure of fibrinogen Caracas II molecules, fibers, and clots were investigated by electron microscopy. Electron microscopy of rotary shadowed individual molecules indicated that most of the alphaC domains of fibrinogen Caracas II do not interact with each other or with the central domain, in contrast to control fibrinogen. Negatively contrasted Caracas II fibers were thinner and less ordered than control fibers, and many free fiber ends were observed. Scanning electron microscopy of whole clots revealed the presence of large pores bounded by local fiber networks made up of thin fibers. Permeation experiments also indicated that the average pore diameter was larger than that of control clots. The viscoelastic properties of the Caracas II clot, as measured by a torsion pendulum, were similar to those of control clots. Both the normal stiffness and increased permeability of the Caracas II clots are consistent with the observation that subjects with this dysfibrinogenemia are asymptomatic.

Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.