Ibaraki Virus,an Agent of Epizootic Disease of Cattle Resembling Bluetongue

Replication of Ibaraki virus was not inhibited by 5-iodo-2′-deoxyuridine, indicating that the virus is an RNA virus. The virus was resistant to ether, chloroform and deoxycholate, sensitive to trypsin, very labile at acidic pH but stable at pH 6.4 or higher, and was resistant to repeated freezing and thawing. The virus was readily inactivated at 56 C or higher, was fairly stable at 37 C, and very stable at 4 C, while it rapidly lost infectivity when stored frozen at —20 C. The virus was readily sedimented by centrifugation at 40 000Xg for 60 min. It readily passed through membrane filters of 200 mμ pore size, passed through 100 μfilters but only with some titer loss and did not through 50 mμ filters. In these tests, the bluetongue virus used as a control behaved in the same manner as Ibaraki virus. These findings provide additional evidence for the similarity of Ibaraki virus to bluetongue virus which had been previously demonstrated on the basis of seasonal incidence, symptomatology and pathology of the diseases caused by these viruses and the behavior of the viruses in cell cultures, embryonated eggs and laboratory animals. The present study, however, provided no evidence for any serological relation between these two viruses. More Information is needed to reach a final decision on the classification of Ibaraki virus, particularly regarding the morphology of the virion, the doublestrandedness of the viral RNA and other basic features.     

Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during andeven after purification. We developed a method to stabilizePPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethyleneglycol at pH 6.5. The protein slowly degraded by itself whenthe stabilizing reagents were removed. Ascorbate and/or H₂O₂accelerated the degradation. The ascorbate-induced degradationwas inhibited by catalase, suggesting that H₂O₂ is generatedthrough reduction of PPO by ascorbate. It is likely that dissolvedoxygen is converted to peroxide through two-electron reductionby the reaction center of PPO, binuclear Cu site, and a Fenton-typereaction occurred on it. This understanding was supported bythe finding that the H₂O₂-induced degradation was inhibitedby metal-chelators as well as by polyphenolic substrate of PPO.Considering the postulated mechanism of the self-degradationof PPO, we re-examined the degradation of the 23-kDa proteinof PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38:179]. The obtained results suggested that the 23-kDa proteintriggers the active oxygen production by the binuclear Cu site,probably as reductant, and receives the radical species preferentiallyto the polypeptide moiety of PPO. (Received April 15, 1999; Accepted July 21, 1999)     

Bovine Diarrhea Virus

Five strains of bovine diarrhea virus were isolated from Japanese cattle using bovine tissue cultures. These are the first isolations of this virus from Japanese cattle to be reported. Of importance is the finding that the new isolates, which are non-cytopathogenic, induce an exaltation of Newcastle disease virus in bovine testicular cell culture. This finding has provided a laboratory tool whereby the assay of the virus and its neutralizing antibody can readily be performed.     

A Markov approach for describing post-fire succession of vegetation

Vegetation dynamics in the coastal area of the Seto Inland Sea region in Japan, where wild fires occur frequently, were described using a stationary Markov model. In this region, vegetation types ofMiscanthus-Pleioblastus grassland,Lespedeza-Mallotus scrub,Pinus-Rhododendron forest andCrassocephalum-Erechtites community have been identified, and these show cyclic succession under the influence of fires. The model uses parameters determining fire frequency and rate of successional change to analyze the effect of variation in these parameters on the areal ratio of each vegetation type at equilibrium and on the time taken for one vegetation type to succeed another (elapsed successional time). The effect of fire frequency differs between hypothetical habitats with high and low productivity. A policy for vegetation management in areas of high and low productivity is proposed. The advantages and limitations of applying Markov models to studies of vegetation succession are also discussed.     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

Mechanisms involved in the cellular uptake of hematoporphyrin by rat hepatoma cells

To clarify the mechanisms involved in the specific uptake of hematoporphyrin by cancer cells, we investigated the interaction of the heme- and/or hematoporphyrin-hemopexin complexes with rat hepatoma dRLh-84 cells. Hemopexin bound to the cells in a saturable, time- and temperature-dependent manner. The cells exhibited 0.55 nmol of binding sites/mg of protein for the heme-hemopexin complex and 0.38 nmol for the hematoporphyrin-hemopexin complex. The dissociation constants (Kd) for the heme-hemopexin and hematoporphyrin-hemopexin complexes were 0.57 and 0.54 microM, respectively. Specific binding of the labeled hemopexin was inhibited by the unlabeled heme- and hematoporphyrin-hemopexin complexes but was unaffected by albumin or neoglycoprotein. Hematoporphyrin bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that hematoporphyrin bound hemopexin was taken up by dRLh-84 cells, via the hemopexin receptors. When the hematoporphyrin-albumin complex was incubated with the cells, the hematoporphyrin-[125I]albumin complex bound to the cells in a time and temperature-dependent manner. Here the binding was not saturated up to 100 micrograms/ml of albumin. The binding of hematoporphyrin-[125I]albumin was partially inhibited by unlabeled albumin and hemopexin. Hematoporphyrin bound to albumin was taken up by the cells at 37 degrees C. Thus, the albumin-dependent uptake of hematoporphyrin by rat hepatoma dRL-84 cells could be differentiated from the hemopexin-mediated uptake of hematoporphyrin.     

Expression and phosphorylation of transferrin receptors in mitogen-activated peripheral blood lymphocytes

Expression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis. The addition of concanavalin A and human serum in combination caused the highest biosynthetic activity. Appearance of the receptor on the cell surface increased in parallel with the degree of the synthesis. Treatment of concanavalin A- and human serum-treated cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a marked stimulation of the phosphorylation of the receptor. Enhancement of phosphorylation occurred within 20 min after the addition of TPA. The density of the receptor on the cell surface slightly increased upon TPA treatment of cells, and the treatment was without effect on iron incorporation from transferrin into the cells. The density of newly synthesized receptor in TPA-treated cells was similar to that in non-treated cells. These results indicated that TPA treatment of mitogen-activated human lymphocytes stimulated the phosphorylation of transferrin receptors, but TPA had no effect on the expression of the receptors thereafter.     

Visible fibrinolysis by endothelial cells: Effect of vitamins and sterols

We have succeeded in corroborating the enhancing effect of vitamin A, vitamin C, sitosterol and fucosterol on the fibrinolytic activity of endothelial cells. The assay system consisted of anin situ dissolution of a fibrin layer coated onto a culture dish, over which endothelial cells were grown in a culture medium containing 10 % serum. The dissolution was enhanced by the addition of these vitamins and phytosterols to the culture medium.To whom correspondence should be addressed.