Mitotic disruption by a novel pyrimidine herbicide, NS-245852, in oat (Avena sativa L.) roots

The effects of a novel pyrimidine herbicide, NS-245852 [2-chloro-6-fluorophenyl-4-(trifluoromethyl)thieno[2,3-d]pyrimidine-2-yl-ketone], on mitosis in oat ( Avena sativa L. cv. Zenshin) root tips were investigated by using light and immunofluorescence microscopy. The root growth was strongly inhibited at 10⁻⁷ mol L⁻¹ of NS-245852, and swollen root tips were induced at 5 × 10⁻⁸ mol L⁻¹. As observed by the use of light microscopy, the herbicide produced disrupted mitosis and large polynucleate cells in the meristematic root tissue. These symptoms were similar to those of mitotic disrupter herbicides. The immunofluorescence microscopy studies of the root tip cells treated for 30 min revealed that spindle fibers and the preprophase band were reduced, although kinetochore fibers and the phragmoplast were not affected. Kinetochore fibers remained as small fluorescence spots, and the phragmoplast disappeared after a 3 h treatment. No microtubule arrays were observed by a longer treatment (longer than 3 h). Among the microtubule arrays, spindle fibers and the preprophase band were found to be the most sensitive to the herbicide, whereas kinetochore fibers were the most resistant. The phragmoplast was intermediate. Thus, the primary action of NS-245852 is the inhibition of polymerization of tubulin into microtubules.     

Inhibitory and promotive allelopathy in rice (Oryza sativa L.)

Studies on allelopathic rice, which inhibits the germination and development of weeds such as barnyardgrass ( Echinochloa spp.) and ducksalad [ Heteranthera limosa (Sw.) Wild.], have been strongly conducted in the USA, the Philippines, Japan, Korea, and other countries since the 1980s. Weed-suppressing rice plants can compete with weeds for light and mineral nutrients by their rapid and thick growth characteristics, and probably also by their allelopathic effects. It was suggested that allelopathic properties were inherited from parents. Different screening methods for finding allelopathic rice simply and rapidly have been proposed. In contrast, stimulative allelopathy in rice, which promotes germination of the dormant and dormancy-awakened seeds of Monochoria vaginalis (Burm. f.) Kunth var. vaginalis , has been investigated by the authors since the 1990s. Rice seeds promoted the germination in the dark and in light under submerged conditions. Our experiments suggested that rice seeds, hulls, plants and straw contain stimulative substance(s) that promote seed germination of M. vaginalis .     

Molecular breeding for virus resistant potato plants

To engineer resistance against potato virus X (PVX), the viral coat protein (CP) gene has been introduced into two potato cultivars. Stable expression of the gene in transgenic clones throughout the growing season has been obtained and resulted in considerably increased virus resistance. With varying frequencies depending on the original cultivar used, true-to-type PVX resistant transgenic clones have been obtained. Since deviant light sprout characteristics were invariably associated with aberrations in plant phenotype, they can be used in procedures to early screen for deviations. Furthermore, it has been possible to unequivocally discriminate between the original untransformed and independent transgenic cultivars. Although no relation has been found between the presence, if any, of the CP of potato virus Y (PVY) or potato leafroll virus (PLRV) in CP gene transgenic potato, appreciable levels of resistance to these viruses has been obtained. This suggests that the mechanism by which a viral CP gene in the potato genome evokes resistance, differs amongst various viruses.     

Structural changes in intramuscular connective tissue during the fattening of Japanese black cattle: effect of marbling on beef tenderization

We investigated changes in structures and mechanical properties of the intramuscular connective tissue during the fattening of Japanese Black steers, using the cell maceration method for scanning electron microscopy. During the early fattening period, from 9 to 20 mo of age, collagen fibrils of the endomysium in longissimus muscle associated more closely with each other, and collagen fibers in the perimysium increased in thickness and their wavy pattern became more regular. These changes were closely related to the increase in mechanical strength of the intramuscular connective tissue and resulted in a toughening of the beef during the period. The shear force value of longissimus muscle decreased after 20 mo of age, concomitantly with the rapid increase in the crude fat content. Scanning electron micrographs of the longissimus muscle dissected from 32-mo-old steers clearly showed that the adipose tissues were formed between muscle fiber bundles, that the honeycomb structure of endomysia was partially broken, and that the perimysium separated into thinner collagen fibers. In semitendinosus muscle, in which the crude fat content was lower (P<.05) than that in longissimus muscle, the structure of the intramuscular connective tissue remained rigid at 32 mo of age. The shear force value of the muscle increased even in the late fattening period, from 20 to 32 mo of age. Thus, the development of adipose tissues in longissimus muscle appears to disorganize the structure of the intramuscular connective tissue and contributes to tenderization of highly marbled beef from Japanese Black cattle during the late fattening period.     

Morphology and size of embryos recovered from superovulated cows.

A total of 691 normal embryos were recovered from 183 superovulated donor cows on the 5th and 6th days after the first insemination, and were examined for their morphology and size in relation to their developmental stage. There was no significant difference in the thickness of the zona pellucida, the diameter of the cell mass, and the overall diameter of the embryos among zygotes, 2-, 4-, 8- and 16-cell embryos, and morulae. In the blastocyst stage, however, the diameter of the cell mass and the overall embryo diameter were significantly greater and the zona pellucida was significantly thinner than in the earlier-stage embryos. The volume of the blastomere significantly decreased from zygote to morula in proportion to the increase in the number of blastomeres. The volume of the cell mass of 2-cell embryos was decreased by about 30% compared with that of zygotes and no increase in the volume of the cell mass was observed during the progression from 2-cell stage to morula. The diameter of the cell mass and the overall diameter of morulae recovered on the 6th day after the first insemination were significantly greater than those of morulae recovered on the 5th day.     

Isolation and monolayer culture of bovine oviduct epithelial cells

Oviduct epithelia obtained from 32 cows were cultured. The oviducts were classified into follicular and luteal phases and divided into ampulla and isthmus regions. The epithelial cells were dissociated by enzyme digestion and cultured in plastic dishes with Dulbecco's modified Eagle's medium/Ham's F12 (1:1) containing 10% calf serum. After enzyme treatment, the epithelial suspension showed free ciliated and non-ciliated cells, and cell mass. The non-ciliated cells contained secretory granules in the cytoplasm. The cell mass was composed of ciliated and secretory cells. The cell mass adhered to the dish within 12-24 hr, while the free ciliated cells attached on Day 2 of the culture. The cells grew into confluent monolayers on Day 4. The cell monolayers contained ciliated and non-ciliated cells. The monolayered non-ciliated cells showed a few secretory granules. When the cells were further cultured without subculturing, ciliary activity diminished on Day 5 and was rarely detected on Day 9. When the cells were subcultured on Day 3, ciliary movement was detected on the monolayers for only 2 days. Cell mass that did not adhere to the dish and remained floating in the medium formed ball-like structures on Day 2. Active ciliary beating was observed on the cells that were cultured in the medium supplemented with 10(-5) and 10(-9) M estradiol-17 beta, however, the ciliary activity diminished on Day 5. No difference in the cell growth was observed between the follicular and luteal phases or between the ampulla and isthmus regions.     

Immunohistochemical analysis of pseudorabies virus spread through neurons innervating the eyeball

Pseudorabies virus (PRV) was inoculated intraocularly into BALB/c mice, and the distribution pattern of cells positive for several neurotransmitters and viral antigens in the eyeball, trigeminal nerve ganglia, and superior cervical ganglia was examined immunohistochemically to clarify the neural route of the virus spread. In the eyeball, substance P (SP)- and calcitonin gene-related peptide (CGRP)-positive cells were detected in the ipsilateral iris and ciliary body, neuropeptide tyrosine (NPY)-positive cells were detected in the choloid membrane, and tyrosine hydroxylase (TH)-positive cells were detected in the ipsilateral inner nuclear layer of the retina; all these cells contained viral antigens. In the superior cervical ganglia, viral antigen-positive cells containing TH or NPY were found at bilateral sites. In the trigeminal nerve ganglia, viral antigen-positive cells containing SP or CGRP were found at bilateral sites. These findings indicated that the SP- and CGRP-positive ganglion cells of the trigeminal nerve ganglia innervating the iris or ciliary body, and the NPY-positive ganglion cells of the superior cervical ganglia innervating the iris, ciliary body, and choroid membrane served as the route for the virus spread. These findings also suggested that dopaminergic neurons, such as the TH-positive retinal cells and TH-positive ganglion cells of the superior cervical ganglia, served as the route for virus spread.