Biological activity of recombinant bovine interferon tau using an Autographa californica nuclear polyhedrosis virus expression system

Bovine interferon (bIFN) tau, which plays a key role in maternal-fetal recognition of pregnancy, was expressed by an Autographa californica nuclear polyhedrosis virus expression system. cDNA coding bIFNtau was derived from cultured trophoblast cells. The recombinant (r) bIFNtau had high antiviral activity (1 x 10 (8) IU/mg) and the molecular weight of rbIFNtau was estimated to be 23 kDa by Western blotting analysis. We investigated the biological effect of rbIFNtau on prostaglandin (PG) F(2alpha) synthesis in cultured bovine endometrial epithelial cells in the presence or absence of oxytocin (OT, 100 nM). rbIFNtau suppressed basal and OT-induced PGF(2alpha) production in a dose-dependent manner (1-1,000 ng/ml). These results showed that biologically active rbIFNtau was produced in the baculovirus expression system, and that rbIFNtau had the ability to suppress the synthesis of PGF(2alpha) from bovine endometrial epithelial cells.     

Heritability estimates of liabilities to digestive diseases in heavy racehorses

Data on 1410 heavy racehorses were taken from the Hokkaido Banei Horse Racing Result Book published during 1997–2000. Heritabilities were estimated using the restricted maximum likelihood method with a full data set of the accumulated prize winnings and the frequency of medical treatment against digestive diseases. Heritability estimates for the same traits were also obtained using a partial data set from affected animals only. In addition, a ‘two classes, one threshold’ model was applied to the full data set for the heritability estimation of liability to the diseases. Heritabilities estimated by restricted maximum likelihood with the full data set were quite low: 0.09 ± 0.02 for the prize winnings and 0.08 ± 0.03 for all three digestive diseases – colic resulting from flatulence, constipation and overfeeding. Low heritabilities were also estimated for each of the three kinds of colic. Medium heritabilities were estimated for colic resulting from constipation (0.30 ± 0.19) and overfeeding (0.46 ± 0.22) using the partial data set taken from the affected animals. Using the threshold model, higher heritabilities from 0.42 ± 0.04 to 0.57 ± 0.03 were estimated for the liabilities to all three digestive diseases and to the individual digestive diseases. These results suggest that liability evaluation of the frequency of medical treatment is insufficient because all non‐affected animals are scored as 0. The high heritabilities estimated by the threshold model with half‐sib relationships should be verified in future studies by comparing them with estimates from other genetic relationships and with realized heritabilities from a small‐scale practical selection program.     

Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.     

Comparisons of Ophiostomatoid fungi associated withTomicus piniperda andT. minor in Japanese red pine

Five species ofOphiostoma, twoLeptographium species and aGraphium species were isolated from two morphologically and ecologically similar bark beetle species,Tomicus piniperda andT. minor, and their infested Japanese red pine (Pinus densiflora) in Yamanashi Prefecture, central Honshu, Japan. An underscribedOphiostoma species andO. minus were isolated mainly fromT. piniperda and its galleries.Ophiostoma canum which was found for the first time in Japan was mainly fromT. minor and its galleries. Specific relationships between the beetles and fungal species are suggested. Contribution No.140, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba. Part of this study was presented at 108th Annual meeting of Japanese Forestry Society, April 2, 1997, Fukuoka, Japan.     

In search of the Kumamoto oyster <Emphasis Type="Italic">Crassostrea sikamea</Emphasis> (Amemiya, 1928) based on molecular markers: is the natural resource at stake?

A cornerstone in conserving wildlife is to resolve taxonomic uncertainties over organisms so that conservationists can define the entity that should be conserved. This is the case for two closely related Crassostrea oysters inhabiting the Ariake Sea (Kyushu, Japan) in sympatry, the kumamoto oyster C. sikamea and Pacific oyster C. gigas, where molecular markers have shed light on their taxonomic separation and species diagnosis. In the past, there was a concern that Kumamoto oysters may be at risk of extinction. However, several field surveys equipped with species-diagnosis molecular markers have provided solid evidence that this perception is incorrect. Nevertheless, previous studies have sometimes given conflicting results, throwing us into confusion about the resource status. Research on natural Kumamoto oysters that the author hopes could help to enhance our understanding of the conservation of this invaluable oyster is reviewed here.

In planta dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission

ABSTRACT Due to the lack of a means to inoculate plants mechanically, the histological dynamics and in planta spread of phytoplasmas have been studied very little. We analyzed the dynamics of plant infection by phytoplasmas, using a technique to infect a limited area of a leaf, nested polymerase chain reaction (PCR), real-time PCR, and immunohistochemical visualization. Following localized inoculation of a leaf of garland chrysanthemum (Chrysanthemum coronarium) by the vector leafhopper Macrosteles striifrons, the onion yellows (OY) phytoplasma spread within the plant from the inoculated leaf to the main stem (1 day postinoculation [dpi]), to the roots and the top leaf (2 dpi), and to other leaves from top to bottom (from 7 to 21 dpi). The populations of the OY phytoplasmas in inoculated leaves and roots increased approximately sixfold each week from 14 to 28 dpi. At 14 dpi, the OY phytoplasmas colonized limited regions of the phloem tissue in both the root and stem and then spread throughout the phloem by 21 dpi. This information should form the basis for elucidating the mechanisms of phytoplasma multiplication and migration within a plant host.     

Immunocytochemical evaluation of epithelial–mesenchymal transition in epithelial tumors of dogs and cats

Epithelial–mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescence-immunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n=22) and cats (n=9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti‐vimentin antibodies. Enzymatic immunohistochemistry (IHC) revealed that 51.6% (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5% (5/8) of those cases. In cats, FICC could detect EMT in 100% (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.     

Possible altitude and temperature limits on pine wilt disease: the reproduction of vector sawyer beetles (Monochamus alternatus), survival of causal nematode (Bursaphelenchus xylophilus), and occurrence of damage caused by the disease

Pine wilt disease is caused by the pine wood nematode [Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle]. In East Asia, an important vector of the nematode is Monochamus alternatus Hope. We determined the tolerance and reproductive ability of sawyer beetles and the nematode to altitude and temperature at elevations between 850 and 1,450 m on Mt. Fuji in Japan. The number of emergent adults decreased markedly along the altitudinal gradient, but the beetle could still reproduce at 1,050 m (8.2 °C annual mean temperature). Beetles with a 2-year life cycle increased rapidly in number with increasing altitude. The pine wood nematode survived through winter at all altitudes tested (850–1,450 m). The beetle population decreased between 950 (9.1 °C) and 1,150 m (8.3 °C). Therefore, the beetle population seems to be stable at 850 m (10.2 °C) and lower altitudes (higher temperatures) but cannot be maintained from 950 (9.1 °C) to 1,150 m (8.3 °C) without constant immigration of beetles from lower altitudes. The beetles could not reproduce at altitudes above 1,150 m (lower than 8.2 °C). From the mean and effective cumulative temperatures, we concluded that the beetle (and its population) can endure temperatures lower than those previously reported. Pine wilt disease also occurred at lower temperatures and higher altitudes than expected. We have summarized the principal strategies for controlling the disease at high altitudes based on these results.