Receptor-induced conformational changes of murine coronavirus spike protein

Although murine coronavirus mouse hepatitis virus (MHV) enters cells by virus-cell membrane fusion triggered by its spike (S) protein, it is not well known how the S protein participates in fusion events. We reported that the soluble form of MHV receptor (soMHVR) transformed a nonfusogenic S protein into a fusogenic one (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In the present study, we demonstrate that soMHVR induces the conformational changes of the S protein, as shown by the proteinase digestion test. A cl-2 mutant, srr7, of the MHV JHM virus (JHMV) was digested with proteinase K after treatment with soMHVR, and the resultant S protein was analyzed by Western blotting using monoclonal antibody (MAb) 10G, specific for the membrane-anchored S2 subunit. A 58-kDa fragment, encompassing the two heptad repeats in S2, was detected when srr7 was digested after soMHVR treatment, while no band was seen when the virus was untreated. The appearance of the proteinase-resistant fragment was dependent on the temperature and time of srr7 incubation with soMHVR and also on the concentration of soMHVR. Coimmunoprecipitation indicated that the direct binding of soMHVR to srr7 S protein induced these conformational changes; this was also suggested by the inhibition of the changes following pretreatment of soMHVR with anti-MHVR MAb CC1. soMHVR induced conformational changes of the S proteins of wild-type (wt) JHMV cl-2, as well as revertants from srr7, srr7A and srr7B; however, a major proportion of these S proteins were resistant to proteinase K even without soMHVR treatment. The implications of this proteinase-resistant fraction are discussed. This is the first report on receptor-induced conformational changes of the membrane-anchored fragment of the coronavirus S protein.     

Abnormalities in cloned mice are not transmitted to the progeny

Cloned animals suffer a wide range of severe fetal and placental malformations. Whether these malformations arise from insufficient epigenetic modifications or mutations has not yet been determined. To address this question, we examined siblings from both cloned XO and XY parents. These parents, which exhibited hypertrophic placentas, increased body weights, and open eyelids at birth, were created from the same ES cell sublines. The siblings from all three cloned pairs showed normal body and placenta weights and no open eyelids at birth. The results clearly showed that the phenotypic abnormalities seen in cloned mice were not transmitted to the progeny, a finding that suggests that abnormalities in cloned mice are responsible for insufficient epigenetic modifications/reprogramming.     

A simple exploratory algorithm for the accurate and fast detection of spontaneous synaptic events

We have developed a program for the fast and accurate detection of spontaneous synaptic events. The algorithm identifies each event of which the slope and amplitude which meet criteria. The significant feature of this algorithm is its stepwise and exploratory search for the onset and the peak points. During the first step, the program employing the algorithm makes a rough estimate of the candidate for a synaptic event, and determines a 'temporary' onset data point. The next step is the detection of the true onset data point and 'temporary' peak data point, which probably exist several points after the temporary onset data point. The third step is a backward search to detect the true peak data point. The final step is to check whether the amplitude of the detected event exceeds the threshold. This stepwise and shuttlewise search allows for the accurate detection of the peak points. Using this program, we succeeded in detecting an increased frequency and amplitude of spontaneous excitatory postsynaptic currents in chick cerebral neurons following the application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In addition, we demonstrated that the program employing the algorithm was able to be used for the detection of extracellular action potentials.     

Elevation of antioxidant potency in mice brain by low-dose X-ray irradiation and its effect on Fe-NTA-induced brain damage

The increase in lipid peroxide levels in mice brain following Fe3+ administration was about 50% of that when 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) was administered. This may be due to excessive oxidation by Fe3+, and was supported by the decrease in activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), Na+,K(+)-ATPase activity and membrane fluidity after Fe3+ administration. Relatively low-dose X-ray irradiation (0.5 Gy) inhibited lipid peroxidation associated with Fe3+ administration and restored the decreased activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity to the levels in the non-Fe(3+)-administered group. In the purine metabolism system, uric acid decreased after Fe3+ administration, which may be due to transient impairment of the system for production of uric acid from xanthine by excessive oxidation by Fe3+. However, 0.5 Gy irradiation inhibited this decrease in uric acid, increasing its level to that in the non Fe(3+)-administrated group. This may be due to factors such as rapid recovery of the activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity after 0.5 Gy irradiation. In addition, since no changes were observed in xanthine and uric acid, increased inosine and hypoxanthine may have advanced to a salvage pathway leading to not xanthine but inosine 5'-monophosphate (IMP).     

Peptide vector for gene delivery with high affinity for phosphatidylserine.

Since phosphatidylserine (PS) is known to translocate to the external face of the plasma membrane when the cell membrane becomes disordered, we decided to focus our attention on PS as a target molecule for gene delivery. In this paper, the novel peptide Td3701 was designed, synthesized, and characterized for its physico-chemico-biological properties. Td3701 simultaneously exhibited both characters as a DNA carrier and a sensor probe for active targeting, which seemed to be triggered by structural changes in the presence of PS. This is a very unique character among nonviral vectors, and it is believed that Td3701 could be used for selective gene delivery.     

Estrous stage- and animal age-independent superovulation in the BrlHan:WIST@Jcl(GALAS) rat.

In most strains of rats, the effects of treatment for the induction of superovulation show major strain differences and are strongly influenced by the stage of the estrous cycle. This study demonstrated, however, that superovulation was easily induced in Wistar strain Brl Han:WIST@Jcl(GALAS) rats by PMSG and hCG administration. To confirm the effects of such treatment, we studied age differences in egg collection efficiency. After superovulation was induced by intraperitoneal administration of 150 IU/kg PMSG and 75 IU/kg hCG given 48 h apart, the mean numbers of oocytes obtained from rats at 4, 8, 12, 20 and 28 weeks of age were 38.9, 33.5, 46.1, 26.9 and 21.3, respectively. No differences caused by the estrous stage at the PMSG administration were observed. In an embryo transfer experiment, fertilized eggs obtained from superovulated rats at each week of age showed equivalent viability until full-term to those from untreated rats. These results suggest that estrous stage-independent superovulation is effective not only in the pre-pubertal stage but also in adult rats.     

Effects of long-term treatment with ethanol on the ultrastructure of the golden hamster parathyroid gland

The ultrastructure of the parathyroid gland in golden hamsters after long-term treatment with ethanol was studied. Male hamsters of experimental groups were given ethanol at the concentration of 7% for 3 and 5 months with food and water freely available. In the ethanol-treated hamsters, the Golgi complexes associated with many prosecretory granules were well developed and many secretory granules were located near the plasma membrane as compared with those of the control animals. Exocytotic events were observed in 5-month-treated animals. These findings suggest that the secretory activity of the parathyroid gland is stimulated after long-term treatment with ethanol.     

Protective effects of 5,6,7,8-tetrahydroneopterin against X-ray radiation injury in mice

The protective effects of 5,6,7,8-tetrahydroneopterin (NH4) against radiation injury in mice were studied. (C57BL/6xA/J)F1 (B6A) mice received a single whole-body irradiation dose of 200, 400, 700 or 800 cGy of X-rays. NH4 (30 mg/kg body weight) or phosphate-buffered saline (PBS) was injected intraperitoneally into irradiated mice 10 min before and after the irradiation and again after 6 h. All mice which received the 800 cGy radiation+PBS died between 8 and 11 days after the treatment. In contrast, those which also received NH4 demonstrated a significantly prolonged survival time and 40% lived more than 5 months. Total numbers of thymocytes and spleen cells on day 5 post-irradiation were dramatically reduced in line with the radiation dose. The survival was significantly enhanced by NH4 in treated mice. The proliferation of spleen cells in mice stimulated by concanavalin A (Con A) or lipopolysaccharide (LPS) was also greater in NH4 treated mice. The immune response of survivors 5 months after 800 cGy+NH4 treatments, against Con A, LPS, allogenic mouse, and sheep red blood cells had essentially recovered to the levels of normal mice. These results indicate that NH4 had an important role in modifying radiation injury.     

Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains

Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli, which binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. We previously demonstrated that Stx induced apoptosis in human renal tubular cell line ACHN cells (Taguchi, T., Uchida, H., Kiyokawa, N., Mori, T., Sato, N., Horie, H., Takeda, T and Fujimoto, J. (1998) Kidney Int. 53, 1681-1688). To study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from ACHN cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomains (DIM). Gb3 was only recovered in DIM and was associated with Src family kinase Yes. Phosphorylation of tyrosine residues of proteins in the DIM fraction increased by 10 min and returned to the resting level by 30 min after the addition of Stx. Since the kinase activity of Yes changed with the same kinetics, Yes was thought to be responsible for the hyperphosphorylation observed in DIM proteins. Unexpectedly, however, all of the Yes kinase activity was obtained in the high density, detergent-soluble fraction. Yes was assumed to be activated and show increased Triton X-100 solubility in the early phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activation by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal transduction.