Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization

Feline leukemia virus (FeLV) is a type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats. The FeLV-induced tumors are thought to be caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. This study was undertaken to enumerate and map the acquired proviral insertions in the genome of a feline thymic lymphoma cell line (FT-1) infected with FeLV. Fluorescence in situ hybridization (FISH) combined with tyramide signal amplification was applied on the chromosome specimen of FT-1 cells and normal cat lymphocytes, with an entire FeLV-A genome used as a probe. Specific hybridization signals were detected from only the metaphases of the FT-1 cells, not from those of normal cat lymphocytes. Statistically based on the Poisson's distribution, at least six loci of chromosomal regions, A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13, and E2p13-p12, appeared to be positive for FeLV integration. Consistently, Southern blot hybridization analysis using an FeLV LTR-U3 probe specific for exogenous FeLV showed the integration of at least six FeLV proviral genomes in FT-1 cells. The cytogenetic technique employed here will provide valuable molecular tags to reveal unidentified tumor-associated genes in FeLV-associated tumor cells.     

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma

Background

CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.

Methods

We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.

Results

Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.

Conclusion

These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.     

Multiplex loop‐mediated isothermal amplification assay for the identification of three major whitefly species in the greenhouse

A multiplex loop‐mediated isothermal amplification (mLAMP) assay was developed for the identification of three species of whitefly, Trialeurodes vaporariorum, Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1) and Mediterranean (MED), major pests in the greenhouse. Each of the specific LAMP primer sets was designed based on the mitochondrial cytochrome oxidase I (mtCOI) gene sequence. The mLAMP reactions using primer mixtures labelled with fluorescent dye were performed at 63°C for 60 min and centrifuged with polyethyleneimine. Thus, T. vaporariorum, MEAM1 and MED were clearly identified by the colour precipitates under UV light. The mLAMP procedure described in this study is cost‐effective and can be performed in the field not only in the laboratory, because this method is a single analysis and does not need a special gene amplification device.     

Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))     

A seed-specific Brassica napus oleosin promoter interacts with a G-box-specific protein and may be bi-directional

Corrigendum

Upstream regulatory sequences from two -conglycinin genes     

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry

Summary The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.     

Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity

Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity.     

Germination ecology of coastal plants in relation to salt environment

Responses of seed germination to salinity were examined using 37 species collected from salt marshes, cliffs, and fore (unstable) and hind (stable) sand dunes along Japanese coasts. For comparison, seed germination of nine inland species was also examined. The soil salinities in salt marshes ranged from 150 to 300 mmol/L NaCl, whereas those in fore and hind dunes ranged from 0 to 150 mmol/L NaCl, with a few exceptions. Cliff soils showed relatively high salinities up to 300 mmol/L NaCl. Ciff and foredune soils that encountered a typhoon and storm showed high salinities >300 mmol/L NaCl. Salt tolerance in seed germination of coastal plants was ordered by comparing the responses of percentage and rate of germination to salinity conditions up to 200 mmol/L NaCl, being in the order of salt marsh>cliff>foredune≅hind dune≅inland. Thse results indicate that salt tolerance in seed germination of coastal plants is closely related to the salinity conditions of their habitats. Germination experiments under favorable conditions showed that a high percentage of the seeds of salt marsh species germinate rapidly, those of diff species germinate slowly and those of foredune species exhibit a low percentage and low rate of germination. It seems that these germination characteristics contribute to the success of germination at the ‘safe site’ and the subsequent survivorship of emerged plants in their natural habitats.