A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100 protein in veterinary diagnostic cytology

The objective of this study was to establish a simple and rapid immunocytochemical technique that can be used in veterinary diagnostic cytology. Air-dried impression smears were collected from canine tumors. Samples of epithelial tumors, mesenchymal tumors, and malignant peripheral nerve sheath tumors and melanomas were used for detection of cytokeratin, vimentin, and S-100 protein, respectively. The labeled streptavidin-biotin system was used in the present study. Optimal fixation was determined using standard immunocytochemical procedures, and acetone fixation was found to be the most effective. Optimal concentrations of primary and secondary antibodies were determined at a preset 5-min incubation. Omission of H(2)O(2) treatment, shortening the time for blocking and labeled-streptavidin incubation, and simplifying washing did not decrease immunopositive intensities or enhance false-positive reactions. The described rapid protocol requires approximately 45min without the use of any special equipment.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

The formation process of button ulcers in pigs experimentally infected with a subgenotype 2.1 isolate of classical swine fever virus

We evaluated the role of classical swine fever virus (CSFV) in the formation of button ulcers in the mucosa of the gastrointestinal tract. Histopathological and immunohistochemical analyses of pigs experimentally infected with a subgenotype 2.1 isolate of CSFV, which was isolated in Japan in 2019, revealed follicular necrosis in the submucosal mucosa-associated lymphoid tissue and herniation of crypts as factors that contribute to the development of button ulcers during CSFV infection. These findings indicate that CSFV induces follicular necrosis and is one of the causative agents of button ulcers in pigs.     

N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.     

Extracellular peroxidase reaction at hyphal tips of white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 and in fungal slime

The distribution of an extracellular peroxidase reaction by white-rot fungus Phanerochaete crassa WD1694 was visualized by peroxidase activity staining. The extracellular peroxidase reaction occurred at the hyphal tips and in the fungal slime filling the gaps between the hyphae. We investigated whether the peroxidase reaction occurred from the hyphal tips or in the slime. The hyphal tips were observed by phase-contrast microscopy, which showed that slime did not exist around the hyphal tips. Time-course observation of hyphal tips showed that peroxidase staining became thick and intense at the tips that did not have fungal slime. Daily observation of the peroxidase staining revealed that the staining was first observed at the hyphal tips. Furthermore, strongly stained hyphae were observed in the stained slime. These results suggested that an active species that oxidizes a peroxidase substrate is first produced at the tips of the hyphae, and then occurs in the slime via diffusion when slime exists around the hyphae. Our results show that the extracellular peroxidase reaction that is important to lignin biodegradation by white-rot fungi occurs directly at the tips of the hyphae and in the slime. Part of this report was presented at the 50th Lignin Symposium, October 19–20, 2005, Nagoya, Japan     

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.     

Construction of a high-density SSR marker-based linkage map of zoysiagrass (<Emphasis Type="Italic">Zoysia japonica</Emphasis> Steud.)

A consensus genetic linkage map with 447 SSR markers was constructed for zoysiagrass (Zoysia japonica Steud.), using 86 F₁ individuals from the cross ‘Muroran 2’ × ‘Tawarayama Kita 1’. The consensus map identified 22 linkage groups and had a total length of 2,009.9 cM, with an average map density of 4.8 cM. When compared with a previous AFLP-SSR linkage map, the SSR markers from each linkage group mapped to similar positions in both maps. Eight pairs of linkage groups from the AFLP-SSR map were joined into eight new groups in the current map. This zoysiagrass consensus map contained 35 SSR markers exhibiting high homology with rice genomic sequences from known chromosomal locations. This allowed synteny to be identified between Zoysiagrass linkage groups 2, 3, 9, 19 and rice chromosomes 3, 12, 2, 7 respectively. These results provide important comparative genomics information and the new map is now available for quantitative trait locus analysis, marker-assisted selection and breeding for important traits in zoysiagrass.     

Glyoxal oxidase supplies hydrogen peroxide at hyphal tips and on hyphal wall to manganese peroxidase of white-rot fungus Phanerochaete crassa WD1694

Peroxidase activity staining localized at hyphal tips of white-rot fungus Phanerochaete crassa WD1694 that was cultivated in a shaken liquid culture containing unbleached kraft pulp was investigated. Manganese peroxidase was detected in culture solution, washing solution of mycelium, and mycelial extract. Glyoxal oxidase was detected only in mycelial extract and was not detected in culture solution. Addition of hydrogen peroxide generated peroxidase activity staining in the culture solution. Addition of catalase resulted in no staining in the culture of P. crassa WD1694, and the addition of methylglyoxal resulted in marked peroxidase activity staining at hyphal tips and on hyphal wall. In an optimized culture, glyoxal oxidase was produced in culture solution. Although the production of glyoxal oxidase and manganese peroxidase had a positive correlation, the secretion and the peak of glyoxal oxidase was observed 3 and 2 days later than those of manganese peroxidase. The N-terminal sequence of purified glyoxal oxidase had very high homology with that of P. chrysosporium. These results elucidated the hydrogen peroxide supply system in lignin biodegradation by white-rot fungi, i.e., while remaining on the hyphal cell wall, glyoxal oxidase provides hydrogen peroxide to manganese peroxidase that had diffused into the culture solution beforehand.     

Fatigue of structural plywood under cyclic shear through thickness II: a new method for fatigue life prediction

For plywood specimens under shear through the thickness, a fatigue life prediction method based on strain energy has been newly developed with the fatigue process and failure criterion applicable to various loading conditions. Once the fatigue process and failure criterion of the plywood specimen were determined by the fatigue data measured under a loading condition other than the square loading waveform, the fatigue life of a specimen under various loading conditions could be predicted easily and accurately by the first cycle loading test. The relationship between stress level and the predicted fatigue life was also similar to that between stress level and the experimentally determined fatigue life. The fatigue life prediction method proposed may be widely applicable to the prediction of the fatigue life of solid wood and wood composites.     

Influence of osmotic pressure on somatic embryo maturation in Pinus densiflora

The effect of an osmoticum, polyethylene glycol (PEG), on somatic embryo production was examined using embryogenic cells of Pinus densiflora. In the basal medium containing 30 μM abscisic acid and 6% maltose, the quality of the embryos formed was poor even though somatic embryos were produced. The addition of PEG with molecular weight of 4000 or 8000 significantly enhanced the development of both the quality and quantity of somatic embryos. Furthermore, higher levels of a constant osmotic pressure with PEG 8000 in a range from about 300 to 450 mmol/kg could remarkably enhance the morphogenesis of somatic embryos and their number of embryos produced. A higher stable osmotic pressure with an appropriate molecular weight of PEG is a key factor for the production of good quality somatic embryos in P. densiflora.