Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Passive impregnation of liquid in impermeable lumber incised by laser

Square lumber specimens of laser-incised Douglas fir (Pseudotsuga menziesii Franco) were treated with steam before dipping. Two types of steam (saturated steam and superheated steam), three steam-injection times (5, 10, and 20 min), four different time intervals (moving time) between steam treatment and dipping (immediate, 3, 10, and 30 min), and four different dipping times (0.5, 1, 3, and 12 h) were used in the study. The maximum absorption was 480 kg/m³ when saturated steam was injected for 20 min and the specimen was immediately dipped into liquid for 12 h. Samples treated with this condition not only absorbed the maximum amount of liquid but also penetrated over 83.4% and 87.3% of the total area along and across the grain, respectively. The optimum conditions were then applied to laser-incised sugi (Cryptomeria japonica D. Don) and Japanese larch (Larix leptolepis Gordon) where the absorption of liquid was 415 and 187 kg/m³, respectively. It was shown that initial moisture content below the fiber saturation point was good for passive impregnation. The absorption of liquid and its distribution in wood indicates that it can be a good preservative treatment method for impermeable woods.     

Effects of Full-Length Kisspeptin Administration on Follicular Development in Japanese Black Beef Cows

Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation.     

Spatiotemporal distribution of barley and wheat plants naturally infected with bacterial black node in fields in western Japan

Bacterial black node caused by Pseudomonas syringae pv. syringae is the most serious bacterial disease of barley and wheat in Japan. The spatiotemporal distribution of barley and wheat plants infected with bacterial black node in fields in 2016–2017 was analyzed using Taylor’s model and Iwao’s model. In Taylor’s model, the sample variance (s ²) of the total and the newly diseased plants at each observation increased with mean plant density (m). In Iwao’s model, although the mean crowding (m*) of total and newly recognized diseased plants increased with m, Taylor’s model fit the data better than did Iwao’s model. Thus, bacterial black node could be explained as a colony expansion model.     

Antifungal activity of a novel compound purified from the culture filtrate of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. O821 against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Rice blast is a devastating fungal disease resulting in major losses to rice crops. Owing to continuous acquisition of resistance by the causal fungus, several fungicide chemicals are no longer effective. Therefore, there is a need to identify natural components and develop new agents to control fungal pathogens. We previously demonstrated that the culture filtrate of Biscogniauxia sp. O821 inhibited infection behavior of Magnaporthe oryzae and subsequent blast lesion formation. In the present study, we isolated a new compound, (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphto[2,3-b]furan-2(3H)-one (HDFO), from the culture filtrate of Biscogniauxia sp. O821 and determined its molecular weight as 248. The HDFO structure was determined by electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopy after purification with column chromatography and high-performance liquid chromatography. The structure of this antifungal compound was similar to that of alantolactone and isoalantolactone. The growth inhibition zone against M. oryzae in presence of HDFO was observed at Rf 0.5–0.6 on a thin layer chromatography plate. HDFO inhibited conidial germination of M. oryzae in a dose-dependent manner (1–200 ppm). Furthermore, blast lesion formation was significantly suppressed by HDFO at over 5 ppm. These results suggest that HDFO from the culture filtrate of Biscogniauxia sp. O821 can protect rice from rice blast disease caused by M. oryzae. This is the first report that HDFO produced by Biscogniauxia sp. can serve as an antifungal compound against M. oryzae.     

Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.     

Fruit rot of sweet pepper caused by <Emphasis Type="Italic">Stemphylium lycopersici</Emphasis> in Japan

A severe rot of postharvest fruits of sweet pepper, a variety of Capsicum annuum, was found in Kagawa Prefecture in southwestern Japan in August 1999. A fungus, isolated repeatedly from the diseased fruits and identified as Stemphylium lycopersici, was demonstrated to be pathogenic to fruits of sweet pepper. The disease was new to Japan, and the fungus was added to the pathogens causing fruit rot of C. annuum.     

Deleterious effects of fungi isolated from paddy soils on seminal root of rice

Soil samples were collected from rice paddies at 22 locations in northeastern Honshu, Japan. In 20 of the samples, seedling growth of rice was improved by soil pasteurization (aerated steaming at 60°C for 30 min), although no typical disease symptoms were observed in the seedlings grown in the untreated soil samples. In most locations, rice seedlings grew better in a potting medium containing root material from plants grown in the pasteurized portion of a soil sample than in a medium with root material from plants grown in the unpasteurized portion of the same sample. The results suggest that microorganisms that restrained rice seedling growth may be common in the soils of rice paddies. Approximately 800 isolates were obtained from seedling roots grown in unpasteurized soils and grouped by cultural and microscopic morphologies. The deleterious effects of 79 isolates from 21 major groups, most of which were soil-dwelling taxa, were examined after direct inoculation of the seminal roots. Isolates of Curvularia sp., Cirrenalia sp., Eppicoccum nigrum, Fusarium graminearum, F. oxysporum, Gliocladium virens, Humicola sp., Penicillium sp., Rhizoctonia oryzae-sativae, Sclerotium hydrophilum, Trichoderma aureoviride, and T. harzianum inhibited root growth, suggesting that deleterious root-infecting fungi were more common in paddy soil than previously thought. These fungi may be involved in the restraint of rice seedling growth.     

Leaf spot of barnyardgrass caused by Exserohilum oryzicola in Japan and the fungal influence on rice,barley, bread wheat,durum wheat,and soybean

Journal of General Plant Pathology - Brown lesions were found on leaves of the weed barnyardgrass in paddy fields in Hiroshima Prefecture in western Japan in June 2017. A fungus, isolated...     

Molecular cloning of canine Mcl-1 gene and its expression in tumor cell lines

The canine Mcl-1 gene was cloned and sequenced. Canine Mcl-1 clone was 2694 base pairs in length and encoded 350 amino acids. The predicted amino acid sequence was 87.7%, 77.1% and 75.7% homologous to predicted human, mouse and rat Mcl-1, respectively. RT-PCR analysis revealed that canine Mcl-1 mRNA was expressed in PBMCs (peripheral blood mononuclear cells), bone marrow cells, MDCK (Madin-Darby canine kidney) and GL-1 (canine B cell leukemia) whereas undetectable in CL-1 (canine T cell lymphoma) cell line.