Measurement of canine blood microparticles by flow cytometry: effect of anticoagulants and staining reagents

Microparticles (MPs) are released from budding plasma membranes into body fluids. The use of flow cytometry for the measurement of MP in canines has not been standardized. In this fundamental study, we compared the effect of anticoagulant agents, such as acid-citrate-dextrose (ACD) and heparin on the measurement of canine MPs in platelet-free plasma (PFP) using flow cytometry. In addition, we used annexin V, carboxyfluorescein succinimidyl ester (CFSE), or calcein tetraacetoxymethyl ester (calcein-AM), and explored the characteristics of the staining reagents in MP detection using flow cytometry. We were able to measure canine MPs in PFP prepared from ACD-anticoagulated blood using flow cytometry, in which the highest positive rate for fluorescent staining was observed when CFSE was used.     

First report of ophidiomycosis in Asia caused by Ophidiomyces ophiodiicola in captive snakes in Japan

Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophiodiicola, which has been affecting wild and captive snakes in North America, Europe, and Australia. We report 12 cases of suspected ophidiomycosis in captive colubrid snakes in Japan. Pathological and microbiological examinations were performed, and the results confirmed the diagnosis of ophidiomycosis in two snakes, which indicated that the remaining sympatrically raised snakes also had ophidiomycosis since they exhibited similar lesions. This is the first report of ophidiomycosis in Asia caused by O. ophiodiicola. To prevent the expansion of ophidiomycosis in the natural environment in Japan, there is a need to evaluate the ophidiomycosis carrier status of imported snakes, the pathogenicity of the infection in native snakes, and the prevalence and distribution of O. ophiodiicola in wild and captive snakes. Measures also must be taken to prevent endemicity globally.     

Effects of GH gene polymorphism and sex on carcass traits and fatty acid compositions in Japanese Black cattle

To investigate the effects of bovine growth hormone (bGH) gene polymorphism on carcass traits and fatty acid compositions in Japanese Black cattle caused by nucleotide substitution of CTG (allele A)/GTG (allele B) at codon 127 and of ACG (allele A and B)/ATG (allele C) at codon 172 of bGH, GH genotypes of 135 cattle were determined using allele specific-multiplex polymerase chain reaction (PCR). Allele A gave greater rib thickness and lower melting point of fat (MP) while allele B gave higher C18:1% ( P  < 0.05). Allele C gave higher C18:1, monounsaturated fatty acid (MUFA), unsaturated fatty acid (USFA) percentages ( P  < 0.05). It also gave lower saturated fatty acid (SFA) percentages, higher MUFA/SFA and USFA/SFA ratios, and lower MP ( P  < 0.05). Interactions of sex and GH alleles were analyzed. In heifers, allele A gave higher carcass weight, daily carcass gain, rib eye area, rib thickness, subcutaneous fat thickness, and BMS while allele B gave greater rib eye area and rib thickness ( P  < 0.05). Allele C gave higher C18:1 ( P  < 0.01), MUFA ( P  < 0.01), USFA percentages ( P  < 0.05) and MUFA/SFA and USFA/SFA ratios ( P  < 0.01), and lower C16:0 and SFA percentages ( P  < 0.05) and MP ( P  < 0.01). GH gene polymorphism affected carcass traits and fatty acid compositions although the effects were more pronounced in heifers.     

Assessment of environmental nitrogen pollution load potential from sewage treatment water in the Tedori River Alluvial Fan Area, Japan

An evaluation of the nitrogen pollution load potential (NPLP) arising from several sewage treatment systems in the Tedori River Alluvial Fan Area was evaluated by determining the removal percentage of nitrogen as a result of sewage treatment and amounts of nitrogen lost from the different treatment systems. These data were applied to the entire area resulting in an estimated total NPLP of 734 t year⁻¹ (10³ kg year⁻¹), of which 548 t year⁻¹ discharges into the Japan Sea and so does not contribute to the pollution of the environment considered here. The residual 186 t year⁻¹ of the NPLP has a high probability of causing groundwater or downstream pollution, with 58% of the NPLP being contributed from septic tanks not connected to public sewage systems. Rural sewage system accounted for about 18% of the NPLP. Material directly flowing into the middle stream of the Tedori River together with sewage water from small companies and untreated sewage water from local family dwellings made up about 3–12% of the NPLP. To improve water quality due to sewage effluent, it is essential to increase the number of connections to public sewage systems from septic tanks.     

Involvement of circadian clock in crowing of red jungle fowls (Gallus gallus)

The rhythmic locomotor behavior of flies and mice provides a phenotype for the identification of clock genes, and the underlying molecular mechanism is well studied. However, interestingly, when examining locomotor rhythm in the wild, several key laboratory‐based assumptions on circadian behavior are not supported in natural conditions. The rooster crowing ‘cock‐a‐doodle‐doo’ is a symbol of the break of dawn in many countries. Previously, we used domestic inbred roosters and showed that the timing of roosters' crowing is regulated by the circadian clock under laboratory conditions. However, it is still unknown whether the regulation of crowing by circadian clock is observed under natural conditions. Therefore, here we used red jungle fowls and first confirmed that similar crowing rhythms with domesticated chickens are observed in red jungle fowls under the laboratory conditions. Red jungle fowls show predawn crowing before light onset under 12:12 light : dim light conditions and the free‐running rhythm of crowing under total dim light conditions. We next examined the crowing rhythms under semi‐wild conditions. Although the crowing of red jungle fowls changed seasonally under semi‐wild conditions, predawn crowing was observed before sunrise in all seasons. This evidence suggests that seasonally changed crowing of red jungle fowls is under the control of a circadian clock.     

Utilization of sweet sorghum bagasse and citric acid for manufacturing of particleboard II: influences of pressing temperature and time on particleboard properties

Development of environmentally friendly particleboard made from sweet sorghum bagasse and citric acid has recently attracted attention. In this study, we investigated the effects of pressing temperature and time on physical properties, such as dry bending (DB), internal bond strength (IB), and thickness swelling (TS) of particleboard. Wet bending (WB), screw-holding power (SH), biological durability, and formaldehyde emission of particleboard manufactured under effective pressing temperature and time were also evaluated. Particleboards bonded with phenol formaldehyde (PF) resin and polymeric 4,4′-methylenediphenyl isocyanate (pMDI) were manufactured as references. Effective pressing temperature and time were 200?°C and 10 min, respectively. It was clarified that DB, IB, and TS satisfied the type 18 requirements of the JIS A 5908 (2003), and were comparable to those of particleboard bonded with PF and pMDI. The WB and SH of particleboard did not satisfy type 18 of JIS. Particleboard manufactured under effective pressing conditions had good biological durability and low formaldehyde emission. Based on the results of infrared spectra measurement, the degree of ester linkages increased with increased pressing temperature and time.     

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.     

Triterpenoids isolated from Zizyphus jujuba inhibit foam cell formation in macrophages

Because foam cell formation in macrophages is believed to play an essential role in the progression of early atherosclerotic lesions in vivo, prevention of foam cell formation is considered to be one of the major targets for the treatment of atherosclerosis. The present study examined the inhibitory effect of 50 crude plant extracts on foam cell formation. Among those crude extracts, Zizyphi Fructus (ZF) and Zizyphi Semen (ZS) extracts significantly inhibited the foam cell formation induced by acetylated LDL. Furthermore, triterpenoids such as oleanonic acid, pomolic acid, and pomonic acid were the major active compounds, and triterpenoids containing a carboxylic acid at C-28 play an important role in the inhibitory effect on foam cell formation in human macrophages. These data suggest that triterpenoids in Zizyphus jujuba , the plant source of ZF and ZS, may therefore be useful for the prevention of atherosclerosis.     

The plasma membrane intactness of root-tip cells is a primary factor for Al-tolerance in cultivars of five species

We investigated the role of the cell wall and plasma membrane (PM) of root-tip cells in Al tolerance in Al-tolerant and Al-sensitive cultivars of five plant species (rice, maize, pea, wheat, and sorghum). No correlation was found between the differences in Al tolerance and the cation exchange capacity of cell walls isolated from root-tips (0–1 em). Preliminary exposure to Al for 1 h was sufficient to inhibit subsequent root re-elongation in an Al-free solution, and the inhibitory effect was more pronounced in the Al-sensitive cultivars than in the Al-tolerant ones. Together with the inhibition of root re-elongation, the PM of the root-tip cells of all the Al-sensitive cultivars was more permeabilized than that of the Al-tolerant cultivars, based on the FDA-PI fluorescence staining technique. Exposure for 30 min to Al treatment at 100 µM significantly increased the PM permeability of protoplasts isolated from the root-tips for the Al-sensitive pea cultivar placed in a moderately hypotonic medium. Protoplasts from root-tip portions of all the Al-sensitive cultivars took up more Al than those of the Al-tolerant ones when treated with 100 pM Al under isotonic conditions for 30 min. The co-existence of DNP or hypotonic conditions led to a larger increase of Al uptake by the protoplasts from Al-sensitive maize cultivars. These results suggest that Al ions rapidly alter the PM of the root-tip portion in the Al-sensitive cultivars, irrespective of plant species, resulting in an increase of the PM permeability.