Effectiveness of small interfering RNA (siRNA) against the Mcl-1 gene in a canine mammary gland tumor cell line

The effect of down-regulation of Mcl-1 expression by small interfering RNA (siRNA) against the canine Mcl-1 gene on apoptosis was investigated by transfecting CF33 (canine mammary gland tumor cell line) with siRNA using cationic liposomes. The siRNA against canine Mcl-1 increased the rate of apoptotic cells and decreased the numbers of viable cells. Further, sequence-specific down-regulation of Mcl-1 expression was measured by real time-PCR and Western blot analysis. The siRNA directed against the Mcl-1 gene reduced both the mRNA and protein expression in the CF33. Our study suggests the importance of Mcl-1 in canine mammary tumors for inducing apoptosis and reinforces using Mcl-1 as a putative therapeutic target in canine mammary gland tumor.     

Characterization of a new viroid strain from hops: evidence for viroid speciation by isolation in different host species

A new viroid was detected in hops cultivated in Akita Prefecture, Japan where it is prevalent in many hops fields. In a survey of hop samples collected during the 1986–2002 growing seasons, the new viroid was present in the major Japanese hop-cultivating areas as early as the 1980s. A single-stranded circular RNA of 368–372 nucleotides that assume a highly basepaired, stable, rod-like secondary structure, shares 93%–98% sequence homology with Apple fruit crinkle viroid (AFCVd) isolated from apple and 85%–87% with Australian grapevine viroid (AGVd) isolated from grapevine. Taking into account the present concept of viroid species, we conclude that the viroid is AFCVd. Circumstantial evidence suggests that AFCVd from apples and hops were endemic in Japan only where cultivation of the two host plants overlapped, thereby strongly supporting the possibility that AFCVd (or an ancestral viroid) was transmitted across the species barrier from apples to hops or hops to apples somewhere in the region. Phylogenetic analysis of AFCVd from hops, AFCVd from apples, and AGVd together with the other members of the genus Apscaviroid revealed that the Akita isolates of AFCVd from hops (AFCVd-hop) formed a cluster that is distinct from AFCVd-apple and AGVd. Accumulation of host-specific sequence variation following their isolation in different host species may be leading to the formation of two viroid species from a common ancestor.     

Expression of hepatitis C virus core protein associated with malignant lymphoma in transgenic mice

Hepatitis C virus (HCV) is a major causative agent for chronic liver diseases leading to hepatocellular carcinoma (HCC) and has also been suggested to be a possible etiologic factor for different lymphoproliferative diseases, including mixed cryoglobulinemia (MC) and B-cell non-Hodgkin's lymphoma (NHL). To understand the roles of HCV core protein in the pathogenesis of HCV related diseases, we produced two lines of the transgenic mice (HC82310 and HC9053) that express the HCV core transgene. One of the lines, HC9053, developed malignant lymphoma (ML, follicular center cell type) with a high frequency (80%) at the ages over 20 months. Hepatocellular adenoma was also observed in this line of transgenic mouse. We demonstrated expression of HCV core protein and mRNA in the liver of transgenic mice, and also detected the core mRNA in the enlarged lymph nodes of the transgenic mice which developed ML. These results suggest that the core protein may play an important role in the development of ML, and that the HC9053 transgenic mice provide suitable models for understanding the mechanism of HCV-related lymphoproliferative diseases.     

Enrichment for isolating Salmonella Choleraesuis and other Salmonella spp. from pigs

The growth of Salmonella Choleraesuis was examined in Rappaport Vassiliadis broth (RV) and Hajna-tetrathionate broth (HTT) at 37 and 42 degrees C. As the enrichment in RV at 37 degrees C was satisfactory for isolating S. Choleraesuis, we used this enrichment for isolation from the samples collected from 15 asymptomatic pigs reared on a S. Choleraesuis contaminated farm. S. Choleraesuis was frequently isolated from six pigs (40.0%) under field conditions. The isolation of other Salmonella serovars than S. Choleraesuis was attempted by using both RV enrichment at 37 degrees C and HTT enrichment at 42 degrees C. Salmonella organisms were isolated from 156 (44.8%) of 348 fecal samples and more frequently with HTT at 42 degrees C (43.4%) than with RV at 37 degrees C (20.9%). If other serovars in addition to S. Choleraesuis are to be surveyed, HTT enrichment should be used in combination with RV enrichment.     

Invasion and colonization of mature apple fruit by Erwinia amylovora tagged with bioluminescence genes

Invasion and colonization of mature apple fruit by a transformant of Erwinia amylovora tagged with bioluminescence genes from Vibrio fischeri was examined. The transformant was deposited on cut surfaces of fruit stems, wounds on the shoulders and calyces, injured fruit-bearing twigs of harvested apple fruit, and cut fruit flesh. After incubation in closed stainless steel or plastic boxes at 25°C, fruit were periodically observed with a two-dimensional luminometer. The presence of the transformant in luminous areas was confirmed by isolating it on selective media. E. amylovora, when deposited in fruit stems: (1) can invade mature as well as immature apple fruit; (2) vertically and horizontally spreads and colonizes along vascular bundles, increasing its population; (3) reaches the calyx end and the flesh just under the exocarp within 3–4 days after inoculation; (4) when deposited on cut fruit flesh, irrespective of its maturity, can easily increase its population and survive 2–4 weeks or more at 25°C; and (5) even at the time of fruit maturation, can migrate within twigs rapidly and reaches the abscission layers between fruit-bearing twigs and fruit stems.     

F-wave latency and F-wave conduction velocity for the tibial nerve in clinically normal dogs

OBJECTIVE: To establish a method of F-wave examinations and to determine values of F-wave conduction velocity (FWCV) and F-wave latency for the tibial nerve of clinically normal dogs. ANIMALS: 21 clinically normal dogs. PROCEDURE: The F-waves were elicited from the interosseous muscles via stimulation of the tibial nerve. The FWCV was determined by using the F-wave shortest value and the surface distance corresponding to the tibial nerve length. Correlation between the smallest latency value of the F-wave and the length of the tibial nerve and between the FWCV and rectal temperature were closely examined. RESULTS: F-wave latency was proportional to the length of the tibial nerve (correlation coefficient, 0.929). Mean +/- SD FWCV was 77.98 +/- 8.62 m/s. Regression equation was as follows: F-wave latency = 2.799 + (0.029 X length of the tibial nerve).The FWCV was increased when the measured rectal temperature was high. Correlation coefficient between FWCV and rectal temperature was 0.665. CONCLUSIONS AND CLINICAL RELEVANCE: In the study reported here, we established a reliable method for clinical evaluation of the F-wave. When assessing nerve conduction velocity, it is essential to measure nerve length along the pathway that the nerve impulse travels. This method of F-wave examination is a useful diagnostic tool for the evaluation of suspected dysfunction of the peripheral nervous system.     

Experimental gizzard erosions in specific-pathogen-free chicks by serotype 1 group I avian adenoviruses from broilers

The pathogenicity of serotype 1 group I avian adenovirus (GIAAV) strains isolated from gizzard erosions for 1-wk-old specific-pathogen-free (SPF) chickens was investigated. In Experiment 1, SPF chickens inoculated orally with a serotype 1 strain (Tokushima2000/GE) of GIAAV isolated from gizzard erosion in broilers were euthanatized and necropsied from 3 to 12 days after inoculation. Clinically, there were no significant signs in the chickens inoculated with Tokushima strain. The gizzards were found macroscopically to be dilated with liquefied contents. The koilin layer of the gizzard exhibited multifocal white lesions with occasional erosions. Gross lesions of the koilin layer (white foci and erosion) appeared at 3 days after inoculation and persisted until 12 days after inoculation. The gross lesions were most severe at 9 days after inoculation. Histologically, multifocal degeneration, necrosis, and loss of the glandular epithelial cells with intranuclear inclusions, liquefactive change of koilin substance, and erosive lesions of koilin layers and surface epithelium of the gizzards were observed. Macrophages increased in the focal areas of the lamina propriabelow the liquefactive koilin substances. Immunohistochemically, intranudear inclusions were positive against GIAAV antigen. Histologic lesions of gizzards were most severe at 7 and 9 days after inoculation, but the frequency of intranuclear inclusions was greatest at 5 days after inoculation. In Experiment 2, the pathogenicities of four strains of serotype 1 GIAAV for 1-wk-old SPF chickens were compared. Three strains isolated from gizzard erosion had similar pathogenicities but one strain from a normal chicken was not pathogenic for chickens. These results suggest that gizzard erosions by serotype 1 GIAAV infection may occur in broiler chickens in Japan.     

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B. cinerea (Bc-19, Bc-45, Bc-682, and Bc-RKR) contained a single base pair mutation in their BcOS1 gene that resulted in an amino acid substitution in the predicted protein. In these resistant isolates, codon 86 of the second repeat, which encodes an isoleucine residue in sensitive strains, was converted to a codon for serine. The mutation of Botrytis field resistant isolates was located on the second unit of tandem amino acid repeats of BcOS1p, whereas the point mutations of the fifth repeat of OS1p confer resistance to both dicarboximides and phenylpyrroles and also osmotic sensitivity in Neurospora crassa. These results suggest that an amino acid substitution within the second repeat of BcOS1p is responsible for phenotypes of field resistant isolates (resistant to dicarboximides but sensitive to phenylpyrroles, and normal osmotic sensitivity) in B. cinerea.     

Identification of the feline CD63 homologue using retrovirus-mediated expression cloning

Recently, we combined a retrovirus-mediated expression cloning with a simple screening method using non-adherent cells and panning [Anal. Biochem. 315 (2003) 138]. In this study, we applied this method to identify the antigen recognized by an uncharacterized monoclonal antibody raised against a feline cell line, and identified it as the feline homologue of CD63. This simple method is useful for characterizing unknown antibodies that recognize cell surface molecules. Furthermore, the monoclonal antibody identified as an anti-feline CD63 antibody will be useful for studying feline molecular function(s).