Construction of recombinant feline herpesvirus type 1 expressing Toxoplasma gondii surface antigen 1.
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Alphaherpesvirinae↗
Biomedical subjects
Y Nishikawa
Publications and source records attributed to Y Nishikawa.
Interfacial and topological measurements of bicontinuous polymer morphologies.
Bicontinuous morphologies are ubiquitous in nature and occur at various length scales. Topological features of two such morphologies arising in an ordered block copolymer at equilibrium and a polymer blend during spinodal decomposition are measured from three-dimensional images. Interfacial curvature, coordination number, and interjunction distance distributions exhibit remarkable similarity in these systems, despite vastly different length scales. A channel coordination of 3 is dominant in both morphologies, and topological measurements such as the Euler-Poincaré characteristic and genus are reported.
Biocompatible Materials↗
Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene.
Neospora caninum infection is the major cause of bovine abortion. To develop a vaccine against N. caninum infection, recombinant vaccinia viruses carrying NcSRS2 and NcSAG1 genes (vv/Nc-p43 and vv/Nc-p36, respectively) were constructed and were tested in a mouse model. Vaccination of dams with vv/Nc-p43 appeared to confer effective protection against vertical transmission to offspring, though that with vv/Nc-p36 only provided partial protection. Moreover, the vv/Nc-p43 vaccination provoked cellular immune responses and antibody production against N. caninum. In conclusion, it is expected that vv/Nc-p43 can be used as an effective live vaccine to prevent vertical transmission of N. caninum in natural hosts.
Animals↗
Protective efficacy of vaccination by recombinant vaccinia virus against Neospora caninum infection.
The recombinant vaccinia viruses expressing the surface protein of Neospora caninum tachyzoite, NcSAG1 or NcSRS2, were constructed. The vaccination with these recombinant viruses could protect effectively the parasite invasion in a mouse model system. The vaccine efficacy of NcSRS2 was higher than that of NcSAG1. The present study indicated that a high level of IgG1 Ab production to parasite is important for clearance of parasite at the early stage of infection and that T cell response has a crucial role for protection against the intracellular infection at the late stage of infection. The recombinant vaccinia viruses might be applicable as vaccine against N. caninum infection in a natural host.
Animals↗
Expression of canine interferon-gamma by a recombinant vaccinia virus and its antiviral effect.
A recombinant vaccinia virus-expressing canine interferon (IFN)-gamma (vv/cIFN-gamma) was constructed. In rabbit kidney (RK13) and canine A72 cells infected with vv/cIFN-gamma, IFN activity was detected in the culture supernatants of both cell types. Canine IFN-gamma was also detected in both cell extracts by Western blot. The activity of the recombinant canine IFN-gamma in RK13 cells was higher than that in A72 cells. The vv/cIFN-gamma could not grow in A72 cells at a low multiplicity of infection, probably due to the antiviral activity of the canine IFN-gamma produced. Although exogenous IFN-gamma did not inhibit the growth of vaccinia virus, addition of anti-canine IFN-gamma serum recovered the growth of the vv/cIFN-gamma on A72 cells in a dose-dependent manner. These results suggest that the growth of vv/cIFN-gamma was inhibited by IFN-gamma produced in a paracrine and autocrine manner. In addition, the recombinant canine IFN-gamma inhibited the multiplication of canine herpesvirus, pseudorabies virus and canine adenovirus type 1 in Madin-Darby canine kidney cells. The antiviral effect of canine IFN-gamma was more effective than that of canine IFN-beta. From the present studies, we concluded the recombinant virus may be a useful suicide viral vector.
Animals↗
Characterization of itch-associated responses of NC mice with mite-induced chronic dermatitis.
To clarify the behavioral and pathological features of spontaneous scratching of NC mice with mite-induced chronic dermatitis, we investigated the spontaneous and pruritogen-evoked scratching of NC mice. Although the frequency of scratching of NC mouse did not increase under specific pathogen-free environment, it gradually and markedly increased from 3 to 6 weeks after transfer to conventional environment. The onset of increase in spontaneous scratching was similar to that of dermatitis development and the elevation of plasma concentration of immunoglobulin E. At chronic stage (16 weeks after environment change), the frequency of spontaneous scratching was roughly parallel to the degree of dermatitis, but not to the plasma concentration of immunoglobulin E. The spontaneous scratching of NC mice with dermatitis was inhibited by distraction and the opioid antagonist naltrexone, suggesting that the scratching is itch-associated response. An intradermal injection of serotonin, but not histamine and substance P, elicited scratching of the injected site. Methysergide and cyproheptadine inhibited the serotonin-induced scratching but not spontaneous scratching. The results suggest that marked elevation of plasma immunoglobulin E is not always the cause of spontaneous itch-associated response of NC mice with dermatitis. Serotonin, histamine and substance P may not play an important role in spontaneous itch-scratch response at a chronic stage.
Animals↗
Interferon-gamma-induced apoptosis in host cells infected with Neospora caninum.
Interferon-gamma (IFN-gamma) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-gamma affects the parasite-infected host cells. The effect of IFN-gamma on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-gamma, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxy-nucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-gamma. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-gamma treated cells following N. caninum infection. Moreover, IFN-gamma treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-gamma induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.
3T3 Cells↗
In the absence of endogenous gamma interferon, mice acutely infected with Neospora caninum succumb to a lethal immune response characterized by inactivation of peritoneal macrophages.
Following infection with Neospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-gamma)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-gamma-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-gamma-deficient mice died within 9 days of infection with N. caninum, whereas those treated with exogenous IFN-gamma lived longer. Although N. caninum invaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-gamma-deficient mice were activated by exogenous IFN-gamma associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-gamma-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-gamma-deficient mice did not. However, in vivo treatment with exogenous IFN-gamma induced up-regulated MHC class II expression in IFN-gamma-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-gamma and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-gamma-deficient mice. The levels of IL-12 in IFN-gamma-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-gamma production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses against N. caninum.
Acute Disease↗
Characterization of Neospora caninum surface protein NcSRS2 based on baculovirus expression system and its application for serodiagnosis of Neospora infection.
The baculovirus expression system has proved to be a useful tool for the production of recombinant proteins. Here we have characterized the Neospora caninum surface protein NcSRS2 produced by two types of the recombinant virus and also have developed an enzyme-linked immunosorbent assay (ELISA) using recombinant NcSRS2 for the serologic diagnosis of Neospora infection. Western blot analysis showed two major protein bands that were detectable in insect cells infected with each recombinant baculovirus, and a lower-molecular-weight protein was detected in culture supernatants from a cell infected with the recombinant virus lacking the hydrophobic C-terminal tail. Analysis of the N-terminal amino acids showed that the secreted NcSRS2 lacked 6 kDa of the N-terminal signal peptide. Moreover, the detergent-soluble protein of insect cells infected with the recombinant baculovirus expressing the full-length NcSRS2 gene was used to develop an ELISA system based on specificity and reactivity to antisera against Toxoplasma gondii, Hammondia heydorni, or N. caninum. Anti-N. caninum mouse, dog, and bovine sera recognized the recombinant NcSRS2 on Western blots. Furthermore, we have shown that the developed ELISA system consistently discriminates indirect fluorescent-antibody test (IFAT)-positive bovine sera against N. caninum from IFAT-negative sera. These results indicate that the ELISA using baculovirus-expressed NcSRS2 can be useful for effective and reliable serodiagnosis of N. caninum infection.
Animals↗
Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.
A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in the B. gibsoni genome. The native P50 protein of B. gibsoni with a molecular mass of 50 kDa was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface of B. gibsoni merozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected with B. gibsoni was developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies to B. gibsoni in dogs.
Amino Acid Sequence↗
Nitric oxide limits coronary vasoconstriction by a shear stress-dependent mechanism.
Increases in shear stress promote coronary vasodilation by stimulating the production of nitric oxide (NO). Whether shear stress-induced NO production also limits vasoconstriction in the coronary microcirculation in vivo is unknown. Accordingly, we measured microvascular diameter and flow velocity in the beating heart along with estimated blood viscosity to calculate shear stress during vasoconstriction with endothelin or vasopressin. Measurements were repeated in the presence of NG-monomethyl-L-arginine (L-NMMA) to inhibit NO production and BQ-788 to block NO-linked endothelin type B receptors. BQ-788 did not augment steady-state constriction to endothelin, suggesting that NO production via activation of this receptor is inconsequential. L-NMMA potentiated constriction to both agonists, particularly in small arteries (inner diameter >120 microm). Shear stresses in small arteries were elevated during constriction and further elevated during constriction after L-NMMA. These observations suggest that NO production limits vasoconstriction in the coronary microcirculation and that the principal stimulus for this governance is elevated shear stress. The degree of shear stress moderation of constriction is heterogeneously distributed, with small arteries displaying a higher degree of shear stress regulation than arterioles. These results provide the strongest evidence to date that shear stress-mediated production of NO exerts a "braking" influence on constriction in the coronary microcirculation.
Animals↗
Identification of 3,4-dihydroxy-2-oxo-butanal (L-threosone) as an intermediate compound in oxidative degradation of dehydro-L-ascorbic acid and 2,3-diketo-L-gulonic acid in a deuterium oxide phosphate buffer.
Dehydro-L-ascorbic acid (DAA), an oxidation product of L-ascorbic acid (vitamin C), is unstable in the neutral and basic pH regions. When DAA was incubated in a phosphate buffer with deuterium oxide (pH 7.4), it was degraded to form the main degradation compound, which was identified as 3,4-dihydroxy-2-oxobutanal (L-threosone). This compound was also formed from diketo-L-gulonic acid (DKG) in a phosphate buffer with deuterium oxide. L-threosone had reducing activity, probably due to its enolization, and is likely to have been involved in the formation of the reducing activity that was observed in aqueous DAA and DKG solutions. As a reactive dicarbonyl compound, L-threosone might also take some role in the cross-linking of tissue proteins that are formed in vivo in the Maillard reaction.
2,3-Diketogulonic Acid↗
Comparison of the growth inhibitory effects of canine IFN-alpha, -beta and -gamma on canine cells infected with Neospora caninum tachyzoites.
The growth inhibitory effects of recombinant canine interferon alpha (IFN-alpha), beta (IFN-beta) and gamma (IFN-gamma) were examined on Madin-Darby canine kidney cells infected with Neospora caninum tachyzoites. The parasite growth was inhibited by all IFNs in a dose-dependent manner. IFN-gamma inhibited the parasite growth with greater efficacy than IFN-alpha or IFN-beta. Moreover, the effect of IFNs on N. caninum growth associated with the suppression of the host cell viability. The present study indicates IFN-alpha and -beta, besides IFN-gamma, play a crucial role for N. caninum growth in host cells.
Animals↗
Phage typing and DNA-based comparison of strains of enterohemorrhagic Escherichia coli O157 from apparently sporadic infections in Osaka City, Japan, 1996.
A marked increase in sporadic cases of enteritis due to enterohemorrhagic Escherichia coli serogroup O157 occurred in Osaka City, Japan, during 1996. To elucidate why the number of cases had increased, the isolates were classified using phage typing, random amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis (PFGE). Fifty-seven percent of the isolates (105/184) belonged to the same phage type (PT-32) and gave the same PFGE pattern; the clone had been isolated during a 3-week period, with a peak on July 15. It was concluded that the majority of the cases identified in July 1996 formed an outbreak, although epidemiological links to a possible common source were not established. The possibility that this outbreak was part of a huge regional outbreak including children at primary schools in Sakai City was discussed.
Bacteriophage Typing↗
6-Deoxy-6-fluoro-L-ascorbic acid: crystal structure and oxidative degradation.
Ascorbic acid and its oxidation products have been implicated in non-enzymatic modification of proteins in aging and diseases of oxidative stress. We have studied the feasibility of using 6-deoxy-6-fluoroascorbic acid (6) for identification of ascorbic acid degradation products by 19F NMR spectroscopy. Crystals of compound 6 from nitromethane belonged to the space group P2(1) with a = 5.547(2), b = 6.769(3), c = 9.302(2) A, beta = 91.80(3) degrees and Z = 2. Atomic coordinates, bond lengths and angles, hydrogen coordinates, anisotropic and isotropic displacement parameters were similar if not identical with those of native ascorbic acid. Similarly, UV properties and oxidation kinetics by CuCl2 at different pH values were essentially identical with ascorbic acid. Using 750 MHz 19F NMR spectroscopy, five to six new fluorinated products were detected after overnight oxidation of 6 with Cu2+, suggesting that 6 may be a powerful and sensitive tool for assessment of its catabolism in vivo.
Ascorbic Acid↗
Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells.
Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), caspase-3-like protease activation (2-4 h), chromatin condensation and DNA ladder formation (3-4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1-3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.
Apoptosis↗
Discovery of novel, potent, and selective small-molecule CCR5 antagonists as anti-HIV-1 agents: synthesis and biological evaluation of anilide derivatives with a quaternary ammonium moiety.
The search for new small-molecule CCR5 antagonists by high-throughput screening (HTS) of the Takeda chemical library using [(125)I]RANTES and CHO/CCR5 cells led to the discovery of lead compounds (A, B) with a quaternary ammonium or phosphonium moiety, which were synthesized to investigate new MCP-1 receptor antagonists. A series of novel anilide derivatives 1 with a quaternary ammonium moiety were designed, synthesized, and tested for their CCR5 antagonistic activity. Through the optimization of lead compounds, we have found N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydr o-2 H-pyran-4-aminium chloride (1r, TAK-779) as a highly potent and selective nonpeptide CCR5 antagonist with a IC(50) value of 1.4 nM in the binding assay. Compound 1r also inhibited the replication of macrophage (M)-tropic HIV-1 (Ba-L strain) in both MAGI-CCR5 cells and PBMCs with EC(50) values of 1.2 and 3.7 nM, respectively. The synthesis and structure-activity relationships of 1r and its related compounds are detailed.
Amides↗
Expression of canine interferon-beta by a recombinant vaccinia virus.
A recombinant vaccinia virus expressing canine interferon (IFN)-beta was constructed (vv/cIFN-beta). In rabbit kidney (RK13) and canine A72 cells infected with vv/cIFN-beta, the recombinant canine IFN-beta was detected in both cell extracts and supernatants, and the IFN activities of the culture supernatants were also detected. Inhibition of N-linked glycosylation by tunicamycin treatment indicated that the recombinant canine IFN-beta was modified by N-linked glycosylation in a different way between RK13 and A72 cells, and that N-linked glycosylation is essential for its secretion. The growth of vv/cIFN-beta at a low multiplicity of infection was inhibited by antiviral activity of canine IFN-beta, indicating that this recombinant virus could be used as a suicide viral vector.
Animals↗