Development of a multiplex RT-PCR detection and identification system for <Emphasis Type="Italic">Potato spindle tuber viroid</Emphasis> and <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis>

We isolated eight polymorphic microsatellite markers for the basidiomycete Armillaria cepistipes and characterised them by analysing 50 isolates representing two geographically distinct populations from Switzerland and the Ukraine. The number of alleles per locus and population varied from one to eight, resulting in 43 alleles over the eight loci and two populations. In both populations, no significant linkage disequilibrium was observed between pairs of loci. Significant (P < 0.05) deviations from Hardy-Weinberg equilibrium were observed at one locus in the Swiss population and at three loci in the Ukrainian population. Of the eight loci developed for A. cepistipes, six were also polymorphic in A. gallica, four in A. ostoyae, two in A. mellea, and one in A. borealis. Beside the potential to be used for population genetic studies on A. cepistipes, these microsatellites thus represent additional molecular markers for three of the four annulated Armillaria species occurring in Europe.     

Roles of oxygen radical production and lipid peroxidation in the cytotoxicity of cephaloridine on cultured renal epithelial cells (LLC-PK1)

To clarify the mechanism of cephalosporin nephrotoxicity, the cytotoxic effects of cephaloridine (CER), a nephrotoxic cephalosporin antibiotic, on the pig kidney proximal tubular epithelial cell line (LLC-PK1) were studied in culture. CER increased the content of hydrogen peroxide and decreased the activity of catalase in the treated cells, followed by an increase in the content of lipid peroxide and decreases in both glutathione peroxidase activity and in the non-protein sulfhydryl content. The levels of NADPH-dependent hydrogen peroxide and superoxide anion production by microsomes prepared from LLC-PK1 cells, and by NADPH-cytochrome P-450 reductase purified from the rat renal cortex were significantly increased by paraquat. The production of these molecules was antagonized by p-chloromer-curibenzoate, an inhibitor of NADPH-cytochrome P-450 reductase. On the other hand, CER did not significantly affect the production of hydrogen peroxide or superoxide anions. These results suggested that the cytotoxic effect of CER on cultured LLC-PK1 cells was due to the increases in hydrogen peroxide and lipid peroxide levels and not microsomal oxygen radical production, and that the mechanism of this cytotoxicity is very different from that of paraquat which induces microsomal oxygen radical production.     

Characterization of lactate dehydrogenase-elevating virus ORF6 protein expressed by recombinant baculoviruses

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity and can replicate only in a subset of mouse primary macrophages in vitro. Because it is difficult to grow and purify sufficient quantities of LDV virions from the primary macrophages, it has been difficult to further characterize LDV envelope proteins. A few expression systems have been reported for structural analysis of the nonglycosylated envelope protein M/VP-2, however, very few studies of the antigenicity of M/VP-2 have been reported. We cloned and expressed the ORF6 gene, which encodes the M/VP-2, as a fusion protein with a polyhistidine metal-binding tag (6×His-tag) in Autographa californica nuclear polyhedrosis virus (baculovirus) under the control of the polyhedrin promoter. In Western blotting analysis, the expressed protein was similar in size to the native M/VP-2 plus 6×His-tag. The usefulness of the baculovirus-expressed LDV ORF6 protein for analysis of the immunogenicity of LDV M/VP-2 was discussed.     

Carbonic anhydrase III in equine tissues and sera determined by a highly sensitive enzyme-immunoassay

A sensitive sandwich enzyme immunoassay (EIA) for measuring equine carbonic anhydrase III (CA-III) was established using a microplate as a solid-phase and peroxidase as a labelling enzyme. The assay can detect concentrations as low as 5 ng/ml using 20 microliters of sample sera. Within-run coefficients of variation obtained using standard equine CA-III were less than 5 per cent. CA-III levels in equine serum ranged from 5 to 50 ng/ml (n = 370), and apparently abnormal levels of CA-III from 100 to 1900 ng/ml (n = 27) were observed. The concentrations of immunoreactive CA-III in the extracts of various equine tissues were also determined; it was present at high concentrations in skeletal muscle and liver and to a much lesser extent in the thymus. Other tissues contained much smaller amounts.     

Comparison of diagnostic tests for the detection of equine infectious anemia antibody

Two diagnostic tests are approved for detecting antibody to equine infectious anemia virus: the agar-gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (ELISA). A total of 420 sera from National Veterinary Services Laboratories check sets were tested with the AGID and competitive ELISA. A 100% correlation was obtained. The AGID and competitive ELISA were further used to test difficult samples with low levels of equine infectious anemia antibody (weak positives). A third test (Western blot) was also used with these weak positive samples to resolve any discordant results.     

X-ray Detection of the Period-Four Cycling of the Manganese Cluster in Photosynthetic Water Oxidizing Enzyme

X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn Kedge showed a period-four oscillation dependent on cycling of the Joliot-Kok's oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S(0)-to-S(1), S(1)-to-S(2), and S(2)-to-S(3) transitions and then reduced upon the S(3)-to-S(0) transition that releases molecular oxygen.     

Anti-Thy-1 Antibody-mediated Complement-dependent Cytotoxicity is Regulated by the Distribution of Antigen,Antibody and Membrane Complement Regulatory Proteins in Rats

Some therapeutic antibodies as anticancer agents exert their effects through the host immune system, but the factors that predict their cytotoxicity, including complement-dependent cytotoxicity (CDC), are unclear. In the present study, we attempted to elucidate some of these factors in a preclinical model. CDC-related mesangiolysis caused by administration of the anti-Thy-1.1 antibody can be studied in the rat anti-Thy-1 glomerulonephritis model, so the model was used in this study. Three animals each were sacrificed at 0.5, 1, 8, 24 and 48 hours after i.v. administration of the anti-Thy-1.1 antibody at 1mg/kg. The distribution of the Thy-1.1 antigen and 2 membrane complement regulatory proteins (mCRPs), Crry and CD55, in three non-treated animals and the distribution of the injected antibody and C3 in the model was studied by immunohistochemistry. In the mesangial cells of the kidney, both expression of the antigen and distribution of the antibody with C3 deposition were observed with weak expression of mCRPs. There was also antigen and antibody distribution in the medullary cells of the adrenal gland and in the lymphocytes of the thymus but no C3 deposition, which was thought to be related to high expression of mCRPs. The antigen was observed in several other organs and tissues without distribution of the antibody. Cell death was only observed in the mesangial cells. These results clearly demonstrate that activation of CDC is regulated by several factors, such as distribution of the target molecule, antibody distribution and the balance among the molecules of the CDC cascade and mCRPs.