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.     

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.     

Purification and properties of haloalkane dehalogenase from Corynebacterium sp. strain m15-3

A haloalkane dehalogenase was purified to electrophoretic homogeneity from cell extracts of a 1-chlorobutane-utilizing strain, m15-3, which was identified as a Corynebacterium sp. The enzyme hydrolyzed C2 to C12 mono- and dihalogenated alkanes, some haloalcohols, and haloacids. The Km value of the enzyme for 1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimum pH for enzyme activity was found to be 9.4, and the optimum temperature was 30 to 35 degrees C. The enzyme was stable for 1 h at temperatures ranging from 4 to 30 degrees C but was progressively less stable at 40 and 50 degrees C.     

Effect of chloramphenicol and lincomycin on chloroplast DNA amplification in greening pea leaves

The light-induced increase in chloroplast DNA was not inhibited by two inhibitors of protein synthesis on 70S polysomes, chloramphenicol and lincomycin, in greening pea leaves. The changes in chloroplast DNA were observed by fluorescence microscopy and measured by hybridization to specific cloned probes. The results suggest that the light-induced increase in chloroplast DNA proceeds without de novo protein synthesis in the chloroplast, in agreement with those with mutants and cultured leaf tissue.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Quantitative fluorescence microscopy on dynamic changes of plastid nucleoids during wheat development

Summary Dynamic change of plastid nucleoids (pt nucleoids) was followed by fluorescence microscopy after staining with 46-diamidino-2-phenyl indole (DAPI). The fluorescence image was quantified with a supersensitive photonic microscope system based on photon counting and image analysis. The results showed that small pt nucleoids located in the center of proplastids in the dry seed increased in size after imbibition and formed highly organized ring structures in the dark, which divided into ca. 10 pieces within 3 days. Corresponding to this morphological change, DNA content of a plastid multiplied 7.5 fold. Total increase in DNA content of pt nucleoids per cell was 34 times as that of dry seed, as plastid multiplied 4.6 times in the average during this period. Upon light illumination small pt nucleoids having basic genome size were separated from divided pt nucleoids, suggesting a relationship with the formation of thylakoid system. The significance of the procedure established in this study is discussed in analysing the dynamic changes of intracellular small genomes.On leave from Department of Biology, Faculty of Science, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

The heterogeneity of arylhydrocarbon hydroxylase in fetal liver microsomes of rats

The characteristic of arylhydrocarbon hydroxylase system in fetal liver microsomes of rat was investigated. NADH-synergistic effect on NADPH-dependent arylhydrocarbon hydroxylase was observed in fetal liver microsomes of rat but not in maternal liver microsomes. NADH-synergistic effect decreased in parallel with the decrease of the ratio of cytochrome b5/cytochrome P-450 in liver microsomes. The cytochrome P-450 in arylhydrocarbon hydroxylase system in fetal liver microsomes of rat seemed to be different from that in offspring liver microsomes in respect of its dependency on cytochrome b5 system for its maximum activity.