Insecticidal and neuroblocking potencies of variants of the imidazolidine moiety of imidacloprid-related neonicotinoids and the relationship to partition coefficient and charge density on the pharmacophore

The pharmacophore of the neonicotinoid insecticide imidacloprid, nitroiminoimidazolidine, was modified to heterocycles such as thiazolidine, pyrrolidine, dihydroimidazole, dihydrothiazole, and pyridone conjugated to nitroimine (=NNO2) or nitromethylene (=CHNO2). Their 6-chloro-3-pyridylmethyl or 5-chloro-3-thiazolylmethyl derivatives were examined for insecticidal activity against the American cockroach by injection and for neuroblocking activity using the cockroach ganglion. Most of the compounds having the neonicotinoidal pharmacophore exhibited insecticidal activity at the nanomolar level, which was enhanced in the presence of synergists, and high neuroblocking activity at the micromolar level. Quantitative analysis for the compounds showed that the neuroblocking potency is proportional both to the Mulliken charge on the nitro oxygen atom and to the partition coefficient log P value. The equation for the insecticidal versus neuroblocking potencies indicated that both potencies are related proportionally with each other when the other factors are the same.     

Development of an enzyme-linked immunosorbent assay to detect an immunomodulatory alpha-D-glucan-protein complex, MPG-1, in basidiomycete Tricholoma matsutake and related processed foods

We previously isolated a novel immunomodulatory alpha-(1,4)(1,6)(1,2)- d-glucan-protein complex (MPG-1) from mycelia of Tricholoma matsutake in basidiomycetes. In the present study, we raised a polyclonal antibody by immunizing rabbits with MPG-1 and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) system to examine the distribution of MPG-1 among edible mushrooms and related processed foods. The system detected MPG-1 quantitatively at concentrations of more than 10 ng/mL, with a coefficient of variation of less than 10% by intra-assay and interassay precision. Analysis with the system of chemically modified MPG-1 suggested that the sugar moiety was mainly involved in the detection. The system detected MPG-1 in the extracts of the fruiting bodies of T. matsutake but not in those of 34 other basidiomycete species. Moreover, a significant amount of MPG-1 was detected in the extracts of their cultured mycelia. The antigenic structure of MPG-1 was relatively stable in terms of pH and temperature. MPG-1 was detected in processed foods supplemented with T. matsutake. These results suggest that MPG-1 is distributed predominantly in T. matsutake species and that the ELISA system can detect it in processed foods supplemented with T. matsutake.     

Isolation and characterization of a novel immunomodulatory alpha-glucan-protein complex from the mycelium of Tricholoma matsutake in basidiomycetes

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.     

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.     

Cardiopulmonary values in dogs with artificial model of caval syndrome in heartworm disease.

Cardiopulmonary values were determined in dogs with an artificial model of heartworm caval syndrome, which was produced by insertion of heartworm-like silicone tubes into the tricuspid valve orifice and right atrium. Fifteen to 25 tubes with some knots were inserted in 6 dogs (knot group), and 7 to 11 tubes (small-number group) or 29 to 37 tubes (large-number group) without a knot in 3 dogs, respectively. After tube insertion, angiographic contrast medium infused into the right ventricle regurgitated to the right atrium in all cases, and the regurgitation was the most severe in the large-number group. On electrocardiographic findings, the atrial and/or ventricular premature beat developed. The height of a- and v-wave of right atrial pressure curves elevated in all groups. The elevation in v-wave was obvious in the large-number group. The pulmonary arterial pressure tended to fall or to elevate slightly, and total pulmonary resistance increased in all groups. The right cardiac output decreased significantly in all cases. The right heart hemodynamics of the model might resemble those in spontaneous cases without disturbed pulmonary circulation.