Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

Myofibrillar proteolysis in chick muscle cell cultures during heat stress

The effect of heat stress on protein oxidation and myofibrillar proteolysis in chick myotubes was investigated. Myotubes were incubated at 37 or 41°C for 6 and 24 h. Protein carbonyl content, as an index of protein oxidation, increased more at 41°C than at 37°C for 6 and 24 h incubations. N^τ‐methylhistidine release as an index of myofibrillar proteolysis also increased more at 41°C than at 37°C for 6 and 24 h incubations. Proteasome activity also increased more under those same conditions. Calpain and cathepsin D but not B + L activities showed a greater increase at 41°C than at 37°C for 24 but not the 6 h incubation. These results indicate that heat stress increases protein oxidation and proteasome activity, resulting in an increase in myofibrillar proteolysis for short‐term incubation and, for long‐term incubation, it increases calpain, proteasome and cathepsin D activities, finally accelerating myofibrillar proteolysis in chick myotubes.     

Identification of domestic cat hepadnavirus from a cat blood sample in Japan

The hepatitis B virus (Hepadnaviridae) induces chronic hepatitis and hepatic cancer in humans. A novel domestic cat hepadnavirus (DCH) was recently identified in several countries, however, the DCH infection status of cats in Japan is unknown. Therefore, we investigated the DCH infection rate of 139 cat samples collected in Japan. We identified one positive blood sample (0.78%) from a 17-year-old female cat with chronically elevated alanine aminotransferase. Phylogenetic analysis demonstrated that the DCH strain identified in this study is genetically different from strains in other countries. Further investigations are required to elucidate the evolution of DCH and the impact of DCH infection on hepatic diseases in domestic cats.     

Change of plasma insulin-like growth factor-1 (IGF-1) concentration with early growth in Japanese beef cattle

The aim of the present study was to examine the change in plasma insulin‐like growth factor‐1 (IGF‐1) concentration with early growth, changes of bodyweight (BW) and relative dairy gain (RDG) in the pre‐ (PRW) and postweaning periods (POW) in Japanese beef cattle, and relationships with metabolites. A total of 33 calves, 22 Japanese black, 6 Japanese shorthorn and 5 of their crossbreed were studied. Insulin‐like growth factor‐1 and metabolite (glucose, triacylglycerol, nonesterified fatty acid) levels in the plasma, from jugular vein blood taken every month, were measured along with BW. Insulin‐like growth factor‐1 in POW increased dramatically with increase of BW (P < 0.05), and the correlation was positive at 0.52 (P < 0.01). Glucose levels correlated significantly with BW, RDG and IGF‐1 (P < 0.01). Metabolic required calorie correlated positively with IGF‐1 (P < 0.01). Also, correlations of BW in POW, with BW and RDG in PRW were positive (P < 0.01). Growth in PRW would be influenced by maternal effects, while active self‐secretion of IGF‐1 in POW might contribute to POW growth. These factors suggested that to increase growth in PRW, maintaining enough maternal effect and IGF‐1 level in POW, was important for establishing better growth after weaning.     

Identification of Petunia hybrida cultivars that diurnally emit floral fragrances

In order to identify genetic resources for breeding fragrant petunias for use as bedding plants, volatile compounds released by day from the flowers of 40 commercial Petunia hybrida cultivars were analyzed using a solid-phase micro-extraction technique coupled with GC–MS. The three cultivars with solid deep-blue flowers that accumulate malvidin in corollas with high tissue pH were found to emit abundant iso-eugenol as the principal floral fragrance. Several other cultivars that emitted considerable amounts of methylbenzoate and/or benzylbenzoate from the flower were also identified. Association between the floral fragrance and the other floral traits such as floral anthocyanin composition and corolla-tissue pH was discussed.     

Effects of oral orbifloxacin on fecal coliforms in healthy cats: a pilot study

The study objective was to determine the effect of oral orbifloxacin (ORB) onantimicrobial susceptibility and composition of fecal coliforms in cats. Nine cats wererandomized to two groups administered a daily oral dose of 2.5 and 5.0 mg ORB/kg for 7days and a control group (three cats per group). Coliforms were isolated from stoolsamples and were tested for susceptibilities to ORB and 5 other drugs. ORB concentrationin feces was measured using high-performance liquid chromatography (HPLC). The coliformswere undetectable after 2 days of ORB administration, and their number increased in mostcats after termination of the administration. Furthermore, only isolates ofEscherichia coli were detected in all cats before administration, andthose of Citrobacter freundii were detected after termination of theadministration. E. coli isolates exhibited high ORB susceptibility[Minimum inhibitory concentration (MIC), ≤0.125 µg/ml]or relatively low susceptibility (MIC, 1−2 µg/ml) with asingle gyrA mutation. C. freundii isolates largelyexhibited intermediate ORB susceptibility (MIC, 4µg/ml), in addition to resistance to ampicillin andcefazolin, and harbored qnrB, but not a gyrA mutation.HPLC revealed that the peaks of mean concentration were 61.3 and 141.0µg/g in groups receiving 2.5 and 5.0 mg/kg, respectively. Our findingssuggest that oral ORB may alter the total counts and composition of fecal coliform, but isunlikely to yield highly fluoroquinolone-resistant mutants of E. coli andC. freundii in cats, possibly because of the high drug concentration infeces.     

Interaction of dinotefuran and its analogues with nicotinic acetylcholine receptors of cockroach nerve cords

To investigate the action of dinotefuran (MTI-446, 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine), a recently developed insecticide, on insect nicotinic acetylcholine receptors (nAChRs), we determined the potencies of the compound and 15 analogues in inhibiting the specific binding of [3H]epibatidine (EPI), a nAChR agonist, and [3H]alpha-bungarotoxin (alpha-BGT), a competitive nAChR antagonist, to the nerve cord membranes of American cockroaches (Periplaneta americana). Racemic dinotefuran inhibited [3H]EPI binding with an IC50 of 890 nM and [3H]alpha-BGT binding with an IC50 of 36.1 microM. Scatchard analysis indicated that the dinotefuran inhibition of [3H]EPI binding was a competitive one. Slight structural modification caused a drastic reduction in potency; only four analogues were found to be equipotent to or more potent than dinotefuran. Chloropyridinyl and chlorothiazolyl neonicotinoid insecticides displayed two or three orders of magnitude higher potency than dinotefuran. There was a good correlation between the IC50 values of tested compounds obtained with [3H]EPI and those obtained with [3H]alpha-BGT. A better correlation was observed between 3-h knockdown activities (KD50) against German cockroaches (Blattella germanica) and IC50 values obtained from [3H]EPI assays than between 24-h lethal activities (LD50) and IC50 values. While the results indicate that dinotefuran and its analogues interact with the ACh-binding site in cockroach nAChRs, it remains to be elucidated why they displayed lower potencies than those expected based on their insecticidal activities.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.