Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.     

Effects of atelocollagen gel containing bone marrow-derived stromal cells on repair of osteochondral defect in a dog

To clarify the contribution of autologous transplantation of mesenchymal stromal cells (MSCs), an atelocollagen gel containing or not containing fluorescently-labeled canine MSCs was transplanted into an osteochondral defect which did not repair spontaneously and the histological repair of the defect was compared. Although an early repair of the cartilage was not observed in either defect, the reproduction of subchondral bone was remarkable in the MSCs-implanted defect. Moreover, in 2 weeks after operation, the implanted MSCs were located in the deeper regions of the defect, suggesting the differentiation of osteoblasts. There was a possibility that the movement of the implanted MSCs was due to an increase in intra-articular pressure from postoperative inflammation.     

Generation of canine dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent antigen-presenting cells that are expected to be therapeutic agents for tumor immunotherapy. In this study, we generated DCs of sufficient number for DC-based immunotherapy from peripheral blood mononuclear cells (PBMC) in dogs. PBMC were cultured in the presence of phytohemagglutinin (PHA). On day 6, large adherent cells with dendrite-like projections were seen, and the number of these large cells with projections increased on day 8. These cells were positive for esterase staining. They expressed MHC class II, CD11b, CD8 and weakly CD4 on their surface. They tended to make contact with lymphocytes under culture conditions. We obtained about 2-5 x 10(6) of DCs from 10 ml of peripheral blood. These DCs phagocytosed HEK-293 cells by overnight co-culturing. These cells generated from PBMC are possible canine DCs and are applicable to clinical trials of DC-based whole tumor cell immunotherapy in dogs.     

A new method for counting of quail leukocytes by flow cytometry

An automatic counting method was developed for fish blood cells using a fluorescent dye, 3, 3-dihexyloxacarbocyanine (DiOC (6)(3)), that selectively stain lipid bilayers in living cells. In the present study, the DiOC(6)(3) method was applied to quail (Cotumix cotumix japonica) blood cells. After quail blood cells were stained with DiOC(6)(3), absolute counts and relative proportions of erythrocytes, granulocytes, monocytes, and lymphocytes plus thrombocytes in whole blood were obtained by means of flow cytometry (FC). The number of each cell types by the FC was in good agreement with those counted microscopically. This method will offer new possibilities for routine blood cell counting for avian medicine.     

An assessment of reproductive and lifetime performances of Kagoshima Berkshire gilts and sows

We investigated the reproductive and lifetime performances of Kagoshima Berkshire gilts and sows. We examined 20 605 parity records of 4419 pigs for 2008 to 2012 on a farrow‐to‐finish commercial farm. The mean parity (± SD) of all animals was 3.0 ± 2.1. For farrowing performance, the highest numbers of total pigs born and pigs born alive were found in sows with parities 5 and 6 and with parity 3–6, respectively (P < 0.05). Regarding weaning and mating performance, sows with parity 2 had the lowest preweaning mortality (P < 0.05). The longest weaning‐to‐first‐mating interval was found in parity 1 pigs, and the interval decreased as parity increased (P < 0.05). Parities 0 and 1 pigs had the lowest farrowing rate and those with parity 4 had the highest farrowing rate (P < 0.05). The mean parity at culling, total number of pigs born alive in a lifetime, and nonproductive days in a lifetime were 5.5 ± 2.93, 49.2 ± 24.72 pigs, and 132.1 ± 83.34 days, respectively. These animals had a lower litter size and fertility that the F₁ crossbred sows mainly used in Japan, but a similar tendency for performance by parity.     

Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.     

Interaction between the helicase domain of the tobacco mosaic virus replicase and a tobacco arginine decarboxylase

In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126K and 183K by ADC.The nucleotide sequence data of pADCF reported in this study is available in the DDBJ/EMBL/GenBank databases under accession number AB110952     

B‐cell intestinal lymphoma with Mott cell differentiation in a 1‐year‐old miniature Dachshund

Abstract: A 1‐year‐old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1‐week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7‐cm‐long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small‐ to medium‐sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott‐like cells contained multiple pale vacuoles that were positive for periodic acid‐Schiff (PAS) in wet‐fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium‐sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS‐positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax‐5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki‐67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax‐5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy‐chain (IgH) gene was observed by PCR testing for lymphocyte–antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B‐cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T‐cell phenotype. The Mott cell morphology also differed from typical mature B‐cell lymphoma types and may be a unique B‐cell lymphoma variant.