Determination of 1-deoxynojirimycin in mulberry leaves using hydrophilic interaction chromatography with evaporative light scattering detection

A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inihibitor present in mulberry leaves (Morus alba and Morus bombysis), by high-performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from an extract of mulberry leaves on a TSKgel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During postcolumn detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products.     

Immunodiagnosis of virulent strains of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) using a monoclonal antibody

Abstract. A virulent strain of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) was used to produce monoclonal antibodies that identified virulent strains of A. hydrophila. Antibodies from a clone, designated as F26P5C8, were found to identify the A. hydrophila serotype I isolates associated with EUS fish, and which were found to be virulent after subsequent inoculation studies. Immunodiagnosis of a large number of A. hydrophila from Australia and Japan showed some additional isolates to be identified by F26P5C8, but the status of their virulence is presently unknown.     

Effect of grain direction on transmittance of 100-GHz millimeter wave for hinoki (<Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis>)

The attenuation coefficients of 100-GHz millimeter waves polarized linearly were measured for cross-cut, quarter-sawn, and flat-sawn boards of hinoki (Chamaecyparis obtusa) that were 0.2–2.0 cm thick. This was done to examine the applicability of free-wave propagation theory for applying electromagnetic waves to wood. It was found that the transmittance of a millimeter wave through the specimen boards was lower when the fiber direction of a board was parallel to the direction of the electric field of the incident wave than when the fiber direction was perpendicular to the electric field, and there was little difference in the transmittance between the tangential and radial directions for the former case. These findings can be quantitatively explained by using propagation theory and the dielectric properties of wood.     

Host immune-related gene responses against highly pathogenic avian influenza virus infection in vitro differ among chicken cell lines established from different organs

Highly pathogenic avian influenza virus (HPAIV) induces acute disease in chickens causing high mortality and morbidity and is a major threat to poultry industries in Southeast Asian countries. The mechanisms of disease manifestation and host innate immune responses against HAPIV in chickens are not well understood. In this study, we examined virus replication and host gene expressions in four chicken cell lines in vitro to elucidate the impact of host innate immune responses against viral replication. It was demonstrated that viral replication efficiencies were different depending on the cell line. The viral replication appeared to be affected by the basal expression of IFN related genes. The expression of immune-related genes against the viral infection also varied in a cell line dependent manner. In non-immune derived cell lines, but not in immune derived cell lines, the expression of the CCL5 and CCL20 genes were induced by HPAIV infection. Reverse genetics HPAIV, with internal genes from avirulent avian influenza, reduced virus replication and affected immune-related gene expression in a cell line dependent manner. These results suggest the possibility that differential immune responses in different cell types in local tissues could modulate the consequences of HPAIV infection in chickens.     

Multiplex PCR and Genescan analysis to detect immunoglobulin heavy chain gene rearrangement in feline B-cell neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.