Fabrication of lead-free piezoelectric (Na(0.5)K(0.5))NbO(3) ceramics by a modified solid-state reaction method.

Sodium potassium niobate, (Na(0.5)K(0.5))NbO(3), fine powder has been successfully synthesized at the low temperature of 550 degrees C through a modified solid-state reaction method, in which urea [CO(NH(2))(2)] plays an important role. High-density (Na(0.5)K(0.5))NbO(3) ceramics could be obtained by conventional sintering of the synthesized (Na(0.5)K(0.5))NbO(3) fine powder with the addition of 0.03 mol% Co(3)O(4) as a sintering additive. The crystal structure, microstructure, and dielectric and piezoelectric properties were characterized. The (Na(0.5)K(0.5))NbO(3) ceramic showed a comparatively saturated P-E hysteresis loop. The (Na(0.5)K(0.5))NbO(3) ceramic also displayed piezoelectricity with a piezoelectric constant d(33) of 126 pC/N and a planar electromechanical coupling factor k(p) of 33%.

Study on the potential of farmland soils as non-point sources of nitrogen and phosphorus in Japan.

The amounts of N and P accumulated in farmland soils of 50 cm depth were equivalent to the amount of chemical fertilizer supplied for 50-70 years. The values of N/P of surface soils in farmlands were 1.0-4.3, lower than expected. The median diameter of soil particles in run-off waters was generally less than 10 microm. The mean values of particulate fractions over 1 microm and over 0.22 microm were 19% for N, 27% for P, and 39% for N, 64% for P respectively. Fine particles of soil containing concentrated phosphorus should be carefully monitored as potential sources related to eutrophication.

Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis.

Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.

Analysis of human IgE epitope of Der f 2 with anti-Der f 2 mouse monoclonal antibodies.

Der f 2 is one of the major mite allergens recognized by human IgE antibodies of allergic patients. Using five anti-Der f 2 mouse monoclonal antibodies, human IgE epitopes of Der f 2 were analyzed. Among them, two monoclonal antibodies 15E11 and 13A4 inhibited the binding between Der f 2 and human IgE antibodies. To determine major IgE epitopes of Der f 2, epitopes for the monoclonal IgG antibodies were analyzed using 43 single site Der f 2 mutants constructed by site-directed mutagenesis. Binding ability of 13A4 and 15E11 was decreased by the amino acid replacement around the C-terminus, and around 73rd, respectively. These results suggest that the C-terminal portion and the central portion around 73rd of Der f 2 were recognized by human IgE antibodies as major epitopes. The location of the putative IgE epitopes on 3-D structure of Der f 2 is also discussed.

A new competitive assay for monoclonal antibodies against phi X174 single stranded DNA.

A new method using L-lysine-conjugated Sepharose 4B as a matrix for coupling competitors is described for analysis of the specificity of monoclonal antibodies against single stranded DNA. In this assay, competitor-coupled Sepharose 4B is first incubated with an antibody, and then the competitor is removed (together with the antibody that is bound to it) by low-speed centrifugation of the reaction mixture. The supernatant containing only unreacted antibody is then analysed by an enzyme-linked immunosorbent assay. The method is simple and does not use radioactive materials. Moreover it is not subject to the data fluctuation that is caused by the binding of competitors to microplates in a commonly used enzyme-linked immunosorbent assay employing high concentrations of competitors.

Recovery of DNA fragments specifically bound by monoclonal anti-DNA antibody.

A method to recover and identify DNA fragments that were specifically bound by a monoclonal anti-DNA antibody has been developed. A mixture of DNA fragments digested by restriction enzymes was first incubated with a murine monoclonal anti-DNA antibody and then reacted with anti-murine immunoglobulin-conjugated Sepharose 4B. The resulting complex was washed to remove unbound DNA by low-speed centrifugation. The bound DNA fragment was released from antibody by alkaline dimethyl sulfoxide solution or extracted by phenol treatment. The recovered DNA was analyzed by electrophoresis on a polyacrylamide gel.

Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-L-lysine.

A new solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of LPS and identification of its serotype with antisera. Since LPS binds poorly to polystyrene microplates, precoating with poly-L-lysine was used before coating LPS on the surface of microplates. The small amount of LPS in complex mixtures (i.e., less than 1 microgram/ml) could be detectable in ELISA. Use of poly-L-lysine with high molecular weight (MW) provided a higher sensitivity than poly-L-lysine with low MW. Precoating with polymyxin B, or poly-L-histidine was less effective in the sensitivity than precoating with poly-L-lysine, but it was still better than no precoating. The newly developed ELISA technique could be also applied for detection of anti-LPS antibodies in sera or for screening of monoclonal anti-LPS antibody.

Action of camptothecin and its derivatives on deoxyribonucleic acid.

Camptothecin, an antitumor alkaloid, induced alkali-labile linkages to supercoiled closed circular DNA. The induction was dependent on the concentration of salt, and the highest level of induction was obtained in the presence of 1.0 M NaCl. The active site was the OH group at the C-20 in the E ring. Camptothecin interacted with adenine base at the C-7 in the B ring which resulted in inhibition of hyperchromicity in heat denaturation of DNA. Replacement of the H group with a substituent whose chain length was longer than -CH2CH3, such as -CH2OCOCH2CH3 or -CH2(CH2)4CO2CH3, caused a loss of potency for the inhibition of hyperchromicity. In the presence of 1.0 M NaCl, camptothecin interacted with superhelical closed circular DNA at 37 degrees. The apparent number of binding sites per base-pair of superhelical DNA was about one hundredth of ethidium bromide according to the equilibrium dialysis experiment. An intercalative interaction was observed with poly(dG-dC) in a 4 M NaCl concentration, but not in 0.1 M NaCl. A similar intercalation was detected in brominated poly(dG-dC) in the physiological concentration of NaCl. Camptothecin seemed to intercalate into the Z-form region which was favorably induced in a negatively superhelical closed circular DNA.

Histological and functional changes in the thyroid glands of mice implanted with hybridomas secreting monoclonal autoantibody against mouse thyroglobulin.

Mouse hybridoma cells secreting monoclonal antibody (mAb) against mouse thyroglobulin were established. The implantation of the hybridomas succeeded to induce high titers of circulating mAb against thyroglobulin in sera of mice. By using the implantation of the hybridomas in mice, the effect of autoantibody on the thyroid glands was studied histologically and functionally. In these mice the thyroid follicles were significantly swollen and warped, whereas there was no infiltration of inflammatory cells. The 125I-uptake in their thyroid glands was markedly decreased. There were no functional changes in control mice implanted with non-secreting P3U1 partner cells. Therefore, it was suggested that high titers of anti-thyroglobulin autoantibody could definitely cause the histological and functional damages in the thyroid glands.

Interaction between SV40 DNA and camptothecin, an antitumor alkaloid.

Camptothecin specifically interacted with closed superhelical circular SV40 DNA during incubation in 1.0 M NaCl at 37 degrees C and induced an alkali-labile linkage in the E strand. No interaction occurred in the reaction mixture containing 0.1 M NaCl, or at 4 degrees C. As camptothecin did not affect linear SV40 DNA, the superhelical structure of DNA appeared to be essential. The site of the alkali-labile linkage induced in SV40 DNA I through interaction with camptothecin was near the origin of replication on the basis of the results of experiments with restriction enzymes. Neither sulfhydryl reagents nor EDTA affected the interaction between camptothecin and SV40 DNA I, so the action of camptothecin is different from those of antitumor antibiotics, bleomycin or neocarzinostatin. Analysis of the s20,0w value of SV40 DNA I after the interaction with camptothecin and the sedimentation profiles of DNA after heating in the reaction mixture indicated that the interaction between camptothecin and SV40 DNA I was different from those of intercalating or alkylating agents such as ethidium bromide and methylmethanesulfonate. Replacement of the OH group at C-20 in the E ring of camptothecin by H-, CH3-, and Cl- resulted in the reduction, in this order, of the potency for interaction with SV40 DNA I to induce an alkali-labile linkage.

Production of tissue factor in CD14-expressing human umbilical vein endothelial cells by lipopolysaccharide.

Production of tissue factor (TF) in response to lipopolysaccharide (LPS) was examined in human umbilical vein endothelial cells (HUVECs) transfected with human CD14 DNA. The expression of CD14 on HUVECs dramatically enhanced the production of TF at a low concentration of LPS in the absence of fetal calf serum (FCS). On the other hand, mock-transfected HUVECs did not respond to even a high concentration of LPS. TF production in CD14-expressing HUVECs was significantly inhibited by anti-CD14 monoclonal antibody. Addition of FCS to the culture of CD14-expressing HUVECs markedly augmented the LPS-induced TF production, whereas only a marginal effect was observed in mock-transfected HUVECs. The findings suggested that the integration of membrane CD14 rendered HUVECs highly sensitive to LPS in the production of TF irrespective of the presence of FCS.

Pure sensory stroke due to pontine haemorrhage.

Two cases of pure sensory stroke due to pontine haemorrhage are reported. Haematomas were located in the dorsolateral tegmentum of the pons, detected by computed tomographic scan. One showed the cheiro-oral syndrome on the left side. The other showed sensory dysaesthesia on the right side of the body, followed by dysaesthesia of cheiro-oral distribution. These cases indicate that the pons can also be one of the lesion sites of pure sensory stroke.

Altered expression of constitutive type and inducible type heat shock proteins in response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.

The expression of heat shock proteins (HSPs) as stress-induced proteins was studied in mice injected with D-galactosamine (D-GalN) and lipopolysaccharide (LPS) as an experimental endotoxic shock model. The expression of constitutive type heat shock protein 70 (HSC70) was significantly reduced in livers of mice injected with D-galactosamine and lipopolysaccharide, while its expression was unaffected in livers of mice injected with D-galactosamine or lipopolysaccharide alone. The expression of other constitutive type heat shock proteins, namely HSP60, HSP32 and HSP25 was also reduced in mice injected with D-galactosamine and lipopolysaccharide. On the other hand, inducible type HSP70 was detected in livers from mice injected with D-galactosamine and lipopolysaccharide, but not in livers from mice injected with D-galactosamine or lipopolysaccharide alone. Simultaneous injection of anti-tumor necrosis factor (TNF)-alpha antibody prevented the liver from reduced expression of constitutive type HSC70, and lead to marked expression of inducible type HSP70 in the liver. Reduced expression of constitutive type HSC70 was also found when D-galactosamine and recombinant TNF-alpha was injected. Therefore, TNF-alpha was suggested to play a critical role on altered expression of constitutive HSC70 and inducible type HSP70 in response of D-galactosamine-sensitized mice to lipopolysaccharide.

Strong adjuvant action of Klebsiella O3 lipopolysaccharide and its inhibition of systemic anaphylaxis.

Immunization with lipopolysaccharide from Klebsiella O3 as an immunological adjuvant did not cause the death of mice in systemic anaphylaxis to bovine serum albumin. On the other hand, most mice immunized with lipopolysaccharide from Escherichia coli O111, Klebsiella O4 and Salmonella minnesota did die. Klebsiella O3 lipopolysaccharide enhanced IgM and IgG antibody response to BSA more markedly than Escherichia coli O111 lipopolysaccharide, while it affected the production of IgE antibody only slightly. therefore, it is suggested that the inhibition of systemic anaphylaxis by Klebsiella O3 lipopolysaccharide adjuvant might be related to its strong adjuvant action on IgM and IgG class antibody production, and that high levels of circulating IgM and IgG antibodies might act as blocking antibodies in the development of IgE-mediated systemic anaphylaxis.

Lipopolysaccharide induces apoptotic cell death of B memory cells and regulates B cell memory in antigen-nonspecific manner.

Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig+ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.

Role of CD86 (B7-2) in triggering of antigen-specific IgE antibody response by lipopolysaccharide.

The role of CD86 in triggering of ascaris extract-specific IgE antibody response by lipopolysaccharide was studied. The simultaneous administration of anti-CD86 antibody with ascaris extract and lipopolysaccharide prevented the production of IgE antibody response to ascaris extract. CD86+ cells were detected in peritoneal cavities and spleens of mice injected intraperitoneally with lipopolysaccharide. CD86+ cells appeared in peritoneal cavities and spleens eight hours after lipopolysaccharide injection, and they were detectable for a week. CD86+ cells in peritoneal cavities and spleens were mainly surface Ig-positive B-cells and some Ig-negative cells. It was suggested that lipopolysaccharide induced the expression of CD86 mainly on B-cells, and that CD86+ cells induced by lipopolysaccharide injection might play an important role as antigen-presenting cells on triggering of ascaris extract-specific IgE antibody response by lipopolysaccharide.

Extracellular matrix components prevent lipopolysaccharide-induced bovine arterial endothelial cell injury by inhibiting p38 mitogen-activated protein kinase.

The effect of extracellular matrix components on lipopolysaccharide-induced vascular endothelial cell injury was studied by using lipopolysaccharide-susceptible bovine aortic endothelial cells. For evaluation of lipopolysaccharide-induced injury, we estimated DNA synthesis and cell detachment of bovine aortic endothelial cells in cultures using extracellular matrix components-coated plastic dishes. Among extracellular matrix components, matrigel almost completely inhibited the reduction in DNA synthesis and the enhancement in cell detachment of bovine aortic endothelial cells in cultures with lipopolysaccharide. The lipopolysaccharide-induced injury was also inhibited by coating with type IV collagen, gelatin, fibronectin, laminin, vitronectin, and heparin sulphate proteoglycan. Extracellular matrix components capable of preventing lipopolysaccharide-induced bovine aortic endothelial cells injury coincidentally inhibited the phosphorylation of p38 mitogen-activated protein kinase in lipopolysaccharide-treated bovine aortic endothelial cells. SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, also prevented the reduction in DNA synthesis and the enhancement in cell detachment of bovine aortic endothelial cells in cultures with lipopolysaccharide. It was therefore suggested that extracellular matrix components might protect bovine aortic endothelial cells from lipopolysaccharide-induced injury through inhibiting the activation of p38 mitogen-activated protein kinase.

Distribution of JC virus DNA in peripheral blood lymphocytes of hematological disease cases.

OBJECTIVE: The distribution of JC virus DNA in peripheral blood was surveyed by the polymerase chain reaction using the late genes as markers. RESULTS: Six out of 52 cases of hematological diseases and one systemic lupus erythematosus case out of 17 cases were positive for JCV DNA. After separation into B and T lymphocytes by a cell sorter, JCV DNA was found in both cell types prepared from adult T cell leukemia and PML patients. CONCLUSION: Only 1 or 2 copies of JCV genome were calculated to exist in a cell based on the time course analysis of PCR. Only B lymphocytes and glial brain cells are known to produce nuclear factors which support the growth of the virus. The result that B lymphocytes contained a copy number of JCV genome similar to T lymphocytes suggests that there is some barrier to viral growth in susceptible B lymphocytes, and that the growth of JCV is different from that of other virulent viruses.

[A case of pontine supranuclear facial palsy].

A 56-year-old hypertensive man suddenly developed difficulty in speaking and numbness in the right hand. On admission, he showed moderate right supranuclear facial palsy and right clumsy hand. Three weeks later, he was discharged with only right supranuclear facial palsy. MRI revealed a small infarction in the middle pons. The lesion was situated in the paramedian borderzone between the base and tegmentum. These findings suggest that supranuclear fibers to the facial nucleus descend as a separate bundle from the main pyramidal tract at the mid-pontine level.